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Stéphane HACQUARD (INRA NANCY)

The secretome of Melampsora larici-populina First results. Stéphane HACQUARD (INRA NANCY). David JOLY (CFL QUEBEC). Nancy, workshop Melampsora , august 2008. I- Introduction. Small secreted proteins ( SSP ) and the establishment of fungal biotrophy.

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Stéphane HACQUARD (INRA NANCY)

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  1. The secretome of Melampsora larici-populina First results Stéphane HACQUARD (INRA NANCY) David JOLY (CFL QUEBEC) Nancy, workshop Melampsora, august 2008

  2. I- Introduction Small secreted proteins (SSP) and the establishment of fungal biotrophy M. larici-populina: obligate biotrophic fungus completely dependant on living plant tissues for growth and development  Specialized infection structure: haustorium Hahn and Mendgen 1997: haustorial ESTs from U. fabae • Early stages of host infection marked by the activation of many genes: PIGs (in Planta Induced Genes) Transport: genes encoding transporters: PIG2- HXT1(Hahn et al, 1997; Voegele et al, 2001) Biosynthesis: genes involved in thiamine biosynthesis (THI1; THI4) (Sohn et al, 2000) Trafficking an signalling process: RTP1 (Kemen et al, 2005)

  3. I- Introduction Catanzariti et al, 2006: haustorial ESTs from M. lini Focus on the secreted proteins  Identification of 21 transcripts encoding Haustorially Expressed Secreted Protein (HESPs)  Among HESPs: AvrL567, expressed in haustoria and trigger specific hypersensitive response-like necrosis that is dependant on the co-expression of the L5, L6, L7 resistance genes (Dodds et al, 2004; Dodds et al, 2006) L9 L5 L6 L7 AvrL567

  4. Clustering of genes encoding secreted proteins Virulence of deletion mutants Unaffected (7) increased (1) Non pathogenic (1) Reduced (3) I- Introduction Kämper et al., 2006:Ustilago maydis secreted proteins  18.6% SP organized in 12 clusters  Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence  SP could trigger defense responses in the host plant (Avr genes) or manipulate host cell structure and fonction to facilitate infection

  5. II- The secretome pipeline: A combination of different prediction softwares What we need to know ? Softwares used  Protein length  Amino acid composition of proteins  Subcellular localization  Presence / Absence of a signal peptide  Protein topology: transmembrane domains PEPSTATS TargetP SignalP TMHMM

  6. II- The secretome pipeline: Why a pipeline ?  Limitation of online prediction program: - number of proteins - length of sequences - number of jobs  Local program: - faster than online services - can apply routine filter to the result  1 command for all computational methods

  7. II- The secretome pipeline: Sequence filter Gene models Prediction of a signal peptide by TargetP SUBCELLULAR LOCALIZATION 2 positive NN scores (Smax and D) plus HMM score by SignalP (Quebec) At least two positive scores by SignalP (Nancy) No transmembrane domains but one domain allowed: N-terminal domain overlaping signal peptide TOPOLOGY LENGTH Gene models  300 amino acids

  8. III- The secretome of Melampsora larici-populina 1-SSP prediction Nancy Ghent Quebec Gene models 16694 16694 16694 Prediction of a signal peptide by targetP 2633 (15,8%) At least two positive prediction methods of a signal peptide by signalP 2125 (12,7%) No transmembrane domains but one domain allowed: N-terminal domain overlaping signal peptide 1848 (11%) Gene models  300 amino acids 961 (5,7%) 797 (4,8%) 818 (4,9%)

  9. III- The secretome of Melampsora larici-populina 1- SSP prediction : • SSP predicted by the 3 pipelines - Nancy, Ghent, Quebec:720 • SSP predicted by 2 pipelines - Nancy, Quebec: 91 -Nancy, Ghent: 52 -Quebec, Ghent: 6 985 putative SSP in Melampsora genome 149 • SSP predicted by only one pipeline - Nancy: 98 - Ghent: 15 -Quebec:3 116

  10. III- The secretome of Melampsora larici-populina 2- Putative SSP homology Among the 985 predicted SSP: Kemen et al. (2005) MPMI 18: 1130-1139  234 contain more than 8 cysteines  %C: all genes: 1,38 / SSP: 2,64  only 247 show homology with nr  171 hits: hypothetical protein • 76 show homology with: • Avirulence genes (AvrL567; AvrM) and Haustorially expressed secreted protein (HESP178, 327, 376, 379, 417, 570, 767, 897, c55, C63, c66, c49) from Melampsora lini.(Dodds et al., 2004; Catanzariti et al., 2006) • Rust transferred protein (RTP1) from Uromyces fabae (Kemen et al., 2005) • Thaumatin, GPI anchored CFEM domain protein, expansin, laccase …  320 show homology with Puccinia

  11. III- The secretome of Melampsora larici-populina 3- SSP families TRIBE-MCL: provides gene family identification based on all-vs-all sequence similarity results.  233 SSP belong to 97 families only composed of secreted proteins(largest:14 members, smallest: 2 members). - large majority of uncharacterized families  151 SSP belong to families composed of both secreted and non secreted proteins - Curation of mispredicted SSPs (NS => S or S => NS)  601 SSP correspond to single gene model

  12. III- The secretome of Melampsora larici-populina 4- SSP annotation August 2008: 401 SSP manually curated (S + D)  114 new or modified gene models (29%)  287 gene models were correctly predicted (71%) EuGene: 103 gene models predicted (35%) fgenesh1: 110 gene models predicted (38%) fgenesh2: 201 gene models predicted (70%)  18 NS => S  Among 155 SSPmanually curatedcorresponding to single model: 45 show homology with others gene models (blastp) or genomic regions (tblastn) => New families

  13. III- The secretome of Melampsora larici-populina 4- SSP annotation Missed models…  One superfamily with more than 30 members  Conserved structure and cysteine residues  Around half were missed by gene predictors

  14. Secreted Non secreted III- The secretome of Melampsora larici-populina 5- SSP clusters 27 clusters of at least 3 SSP separeted by no more than 3 non secreted proteins. Scaffold 11: Homology with HESP-417 Homology with HESP-417 Hypothetical protein Hypothetical protein Hypothetical protein Homology with HESP-417 Scaffold 31: Cutinase Homology with RTP1 Cutinase 1kb

  15. III- The secretome of Melampsora larici-populina 6- SSP expression data Melampsora cDNA libraries  Urediniospores library (Sanger ESTs): - M. larici-populina spores and germlings (JGI, Nancy) 52,269 ESTs  Infected poplar leaves library (454 pyrosequencing ESTs): - M. larici-populina 98AG31 infected ‘Beaupré’ leaves (compatible interaction) at 4 dpi and 7 dpi (50/50mixed) 4679 ESTs Melampsora 5533 ESTs poplar

  16. III- The secretome of Melampsora larici-populina 6- SSP expression data 985 SSP: 323 supported by EST (33%) • 58only supported by spores ESTs (sanger) • 97 supported by both spores and infected leaves ESTs (Sanger and 454)  168 only supported by infected leaves ESTs (454)  58 SSP only supported by spores ESTs 19 homologies with nr, 5 hits against known proteins

  17. III- The secretome of Melampsora larici-populina 6- SSP expression data  97 SSP supported by both spores and infected leaves ESTs 51 homologies with nr, 17 hits against known proteins - Most ESTs belong to spores: - Most ESTs belong to infected leaves:

  18. III- The secretome of Melampsora larici-populina 6- SSP expression data  168 SSP only supported by infected leaves ESTs 47 homologies with nr, 15 hits against known proteins

  19. 160 140 120 100 80 60 40 20 0 Sp 2 6 12 24 48 96 168 Relative expression of M. larici-populina genes (2-∆Ct) to ElF and Tub reference genes by RT-qPCR. Time (hours post-inoculation) 160 140 120 100 80 60 40 20 0 168 Sp 2 6 12 24 48 96 168 Sp 2 6 12 24 48 96 Sp 2 6 12 24 48 96 168 Sp 2 6 12 24 48 96 168 140 120 140 120 120 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 180 160 140 120 100 80 60 40 20 0 Sp 2 6 12 24 48 96 168 III- The secretome of Melampsora larici-populina 6- SSP expression data • Effectors previously described in Pucciniales are also expressed in M. larici-populina. • Specific transcripts profiles are observed at the different stages of the infectious process. • Interestingly, two genes encoding RTP1 homologues have distinct expression profiles.

  20. III- The secretome of Melampsora larici-populina 7- SSP evolutionary constraints Positive selection screen on an EST data set…  Application to whole genome?

  21. V- Conclusions 985 putative SSP in Melampsora genome (5.8%)  33% supported by ESTs  Most of SSP expressed during infection process  Homology with effectors previously described in Pucciniale  660 SSP specific of Melampsora Validation data: Biological approch - NimbleGen micro-array - RT-qPCR - immunolocalization

  22. Acknowledgment NANCY: Emilie TISSERANT Benoit HILSELBERGER Marie Pierre OUDOT-LE SECQ Sébastien DUPLESSIS Francis MARTIN GHENT: Yao-Cheng LIN Yves van De Peer QUEBEC: Nicolas FEAU Richard HAMELIN JGI sequencing and annotation team

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