1 / 22

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.

dillan
Télécharger la présentation

Stéphane HACQUARD (INRA NANCY)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related