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

2. I- Introduction 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) 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

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) AvrL567 L9 L5 L6 L7

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

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

6. 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 II- The secretome pipeline:

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

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

9.  SSP predicted by the 3 pipelines - Nancy, Ghent, Quebec: 720  SSP predicted by 2 pipelines - Nancy, Quebec: 91 - Nancy, Ghent: 52 - Quebec, Ghent: 6  SSP predicted by only one pipeline - Nancy: 98 - Ghent: 15 - Quebec: 3 III- The secretome of Melampsora larici-populina 1- SSP prediction : 985 putative SSP in Melampsora genome MelampsoraPucciniaLaccaria Gene models166942056720613 SSP  300aa 985 (5,8%)1219 (5,9%)864 (4,2%) 149 116

10. III- The secretome of Melampsora larici-populina Among the 985 predicted SSP:  171 hits: hypothetical protein  76 show homology with:  234 contain more than 8 cysteines  320 show homology with Puccinia 2- Putative SSP homology Kemen et al. (2005) MPMI 18: 1130-1139 - 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 …  %C: all genes: 1,38 / SSP: 2,64  only 247 show homology with nr

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).  151 SSP belong to families composed of both secreted and non secreted proteins  601 SSP correspond to single gene model - large majority of uncharacterized families - Curation of mispredicted SSPs (NS => S or S => NS)

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

15. 6- SSP expression data III- The secretome of Melampsora larici-populina 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. 6- SSP expression data III- The secretome of Melampsora larici-populina 985 SSP: 323 supported by EST (33%)  58 only 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  97 SSP supported by both spores and infected leaves ESTs 6- SSP expression data - Most ESTs belong to spores: 51 homologies with nr, 17 hits against known proteins - 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. III- The secretome of Melampsora larici-populina 6- SSP expression data 0 20 40 60 80 100 120 Sp2612244896168 0 20 40 60 80 100 120 140 160 180 Sp2 6 12 24 4896 168 0 20 40 60 80 100 120 140 Sp2612244896 168 0 20 40 60 80 100 120 140 160 Sp2612244896168 0 20 40 60 80 100 120 140 Sp2612244896 168 0 20 40 60 80 100 120 140 160 Sp2612244896168 Relative expression of M. larici- populina genes (2- ∆Ct ) to ElF and Tub reference genes by RT-qPCR. Time (hours post-inoculation)  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 HGGLocBLASTX against Fungi nrE-valueCysω 205NSNo hitND2.68 254NSNo hitND2.59 1278NS ref|XP_001211022.1| conserved hypothetical protein [Aspergillus terreus] 3e-07ND1.88 28SNo hit01.38 92S gb|AAS45284.1| proline-rich antigen [Chrysosporium lucknowense] 4e-0961.43 729S gb|EAT81533.2| hypothetical protein [Phaeosphaeria nodorum] 7e-1181.32 747S ref|XP_757360.1| hypothetical protein [Ustilago maydis] 1e-0441.95 4191SNo hit41.57 5606SNo hit61.76 5610SNo hit64.31 5617SNo hit01.23 5624SNo hit81.93 6067S ref|XP_758577.1| hypothetical protein [Ustilago maydis] 6e-06101.27 Positive selection screen on an EST data set…  Application to whole genome?

21. V- Conclusions 985 putative SSP in Melampsora genome (5.8%)  Homology with effectors previously described in Pucciniale  33% supported by ESTs  660 SSP specific of Melampsora  Most of SSP expressed during infection process 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 JGI sequencing and annotation teamQUEBEC: Nicolas FEAU Richard HAMELIN