stéphane hacquard (inra nancy)

Stéphane HACQUARD (INRA NANCY)

The secretome of Melampsora larici-populina First results

Nancy, workshop Melampsora, august 2008

David JOLY (CFL QUEBEC)

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

QuickTime™ et undécompresseur TIFF (LZW)

sont requis pour visionner cette image.

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)


sont requis pour visionner cette image.AvrL567

L9 L5 L6 L7

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 proteins Virulence of deletion mutants


sont requis pour visionner cette image. 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

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:

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


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


2 positive NN scores (Smax and D) plus

HMM score by SignalP(Quebec)

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%)

Ghent Quebec

797 (4,8%) 818 (4,9%)

1-SSP prediction

16694 16694

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


1- SSP prediction :

985 putative SSP in Melampsora genome

Melampsora Puccinia Laccaria

Gene models 16694 20567 20613

SSP 300aa 985 (5,8%) 1219 (5,9%) 864 (4,2%)

149

116


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


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)


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


4- SSP annotation

Missed models…

One superfamily with more than 30 members

Conserved structure and cysteine residues

Around half were missed by gene predictors


5- SSP clusters

Secreted Non secreted

Scaffold 31:

Scaffold 11:

Cutinase 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.

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



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)

source # readsGene model

SSP length # C Blastp nr

sanger 274 72276 N, G, C 290 5 No hits foundsanger 192 95785 N 67 0 No hits foundsanger 167 55041 N, G, C 191 3 No hits foundsanger 84 70656 N, C 295 2 gb|ABS86600.1| rds1p-like protein [Melampsora medusae f. sp. deltoidis] e-100 369sanger 82 72205 N, G, C 259 6 ref|XP_001801287.1| hypothetical protein SNOG_11034 [Phaeosphaeria nodorum SN15] 3e-12 75sanger 26 72535 N, G 298 4 No hits foundsanger 9 67341 N, G, C 173 6 No hits foundsanger 8 33903 N 185 0 ref|XP_758980.1| hypothetical protein UM02833.1 [Ustilago maydis 521] 1e-24 115sanger 8 74706 N, G, C 208 4 No hits foundsanger 8 55804 N, G, C 216 0 ref|XP_001393628.1| hypothetical protein An09g03120 [Aspergillus niger] 2e-12 75

sanger 3 74496 N, G, C 132 5 gb|ABS86322.1| hesp-417-like protein [Melampsora medusae f. sp. tremuloidis] 2e-35 150sanger 2 71126 N, G, C 178 15 gb|ABB96284.1| hesp-C49 [Melampsora lini] 7e-75 282sanger 2 49700 N, G, C 255 6 ref|YP_001637101.1| Alpha-amylase [Chloroflexus aurantiacus J-10-fl] 2e-65 252sanger 1 76068 N, G, C 185 8 emb|CAA66278.1| thaumatin-like protein [Triticum aestivum] 9e-18 92

58 SSP only supported by spores ESTs

19 homologies with nr, 5 hits against known proteins


97 SSP supported by both spores and infected leaves ESTs


- Most ESTs belong to spores:




sanger_454 326_2 72911 N, G, C 295 12 No hits foundsanger_454 246_2 70937 N, G, C 285 7 ref|XP_569376.1| hypothetical protein [Cryptococcus neoformans var. neoformansJEC21] 6e-23 111sanger_454 178_2 71932 N, G, C 247 4 gb|EAU84698.1| predicted protein [Coprinopsis cinerea okayama7#130] 5e-20 101sanger_454 65_1 70587 N, G, C 207 0 gb|ABB96272.1| hesp-379 [Melampsora lini] 2e-59 231sanger_454 36_1 78206 N, G, C 231 4 ref|XP_569628.1| hypothetical protein [Cryptococcus neoformans var. neoformansJEC21] 3e-29 131

sanger_454 11_6 71404 N, G, C 128 5 gb|ABB96273.1| hesp-417 [Melampsora lini] 2e-47 190sanger_454 95_27 116059 N, G, C 287 2 ref|XP_567927.1| nucleus protein [Cryptococcus neoformans var. neoformans JEC21] 6e-19 98



454_sanger 2366_4 75678 N, G, C 147 0 No hits found454_sanger 224_4 74588 N, C 177 10 gb|EDR13473.1| predicted protein [Laccaria bicolor S238N-H82] 1e-30 135454_sanger 44_1 84974 N, G, C 152 6 No hits found454_sanger 27_1 117675 N, G, C 287 4 ref|XP_566486.1| disulfide-isomerase precursor [Cryptococcus neoformans var.neoformans JEC21] 2e-43 179454_sanger 67_3 74599 N, G, C 129 4 No hits found

454_sanger 20_3 70546 N, G, C 164 9 gb|ABV46587.1| GPI anchored CFEM domain protein [Monacrosporium haptotylum] 2e-08 61454_sanger 22_9 71399 N, G, C 132 5 gb|ABS86322.1| hesp-417-like protein [Melampsora medusae f. sp. tremuloidis] 3e-35 150454_sanger 9_4 72001 N 189 1 gb|EDR03200.1| GTP-binding protein sar1 [Laccaria bicolor S238N-H82] 4e-96 353454_sanger 12_5 106888 N, G, C 220 1 ref|XP_571865.1| cyclophilin [Cryptococcus neoformans var. neoformans JEC21] 3e-66 254454_sanger 16_11 118503 N, G, C 153 6 dbj|BAF46900.1| glycopeptide [Phanerochaete chrysosporium] 1e-09 65454_sanger 6_2 42266 N, C 85 0 gb|ABB96277.1| hesp-767 [Melampsora lini] 1e-44 181

- Most ESTs belong to infected leaves:





454 379 103712 N, G, C 135 3 No hits found454 92 105045 N, G, C 123 2 No hits found454 78 107730 N, G, C 182 4 No hits found454 50 111920 N, G, C 155 4 No hits found454 42 63414 G, C 209 4 No hits found454 36 107547 N, G, C 155 1 No hits found454 35 106106 N, G, C 198 14 No hits found454 32 69494 N, G, C 178 2 No hits found454 30 63784 N, G, C 194 8 dbj|BAE57901.1| unnamed protein product [Aspergillus oryzae] 2e-14 82454 25 69785 N, G, C 166 1 No hits found

454 22 85787 N, G, C 266 16 gb|EDR10333.1| thaumatin-like protein [Laccaria bicolor S238N-H82] 2e-71 272454 20 86842 N, G, C 167 8 gb|ABB96268.1| hesp-178 [Melampsora lini] 2e-05 51454 16 79324 N, G, C 186 8 emb|CAA66278.1| thaumatin-like protein [Triticum aestivum] 7e-18 93454 13 107475 G, C 232 7 gb|ABS86408.1| rust transferred protein [Melampsora occidentalis] 1e-75 286454 12 91058 N, G, C 152 5 ref|XP_570934.1| FK506 binding protein 2 [Cryptococcus neoformans var. neoformansJEC21] 1e-43 177454 10 39287 N, C 165 2 gb|AAF87492.1| AF232039_1 Cro r I [Cronartium ribicola] 1e-21 105454 8 102766 N, C 151 4 ref|XP_570234.1| vacuole protein [Cryptococcus neoformans var. neoformans JEC21] 6e-24 112454 6 53594 N 179 2 ref|NP_001012886.1| ADP-ribosylation factor-like 10B [Gallus gallus] 5e-60 233454 6 37534 N, G, C 77 8 gb|ABB96283.1| hesp-C66 [Melampsora lini] 8e-15 82454 4 90815 G 248 3 gb|AAR37848.1| cytochrome P450 family protein [uncultured marine bacterium 443] 3e-13 79454 3 109896 N, G, C 121 5 dbj|BAD11818.1| riboflavin aldehyde-forming enzyme [Lentinula edodes] 4e-17 90454 1 31952 N, G, C 79 1 gb|ABB96270.1| hesp-327 [Melampsora lini] 1e-19 98454 1 37332 N, G, C 138 1 gb|ABB96271.1| hesp-376 [Melampsora lini] 2e-28 127454 1 101227 N, G, C 229 8 ref|XP_001259883.1| extracellular cellulase CelA, putative [Neosartorya fischeri NRRL181] 3e-43 178454 1 58404 N, G, C 263 1 ref|YP_001180662.1| Phosphoglycerate mutase [Caldicellulosiruptor saccharolyticus DSM8903] 3e-13 79

168 SSP only supported by infected leaves ESTs




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Sp T2 T6 T12 T24 T48 T96 T168

Mlp-Hesp417-like

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Sp 2 6 12 24 48 96 168

020406080

100120140160180

Sp T2 T6 T12 T24 T48 T96 T168

Mlp-Rtp1sc32-like

020406080

100120140160180

Sp 2 6 12 24 48 96 1680

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Sp T2 T6 T12 T24 T48 T96 T168

Mlp-AvrP123-like

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Sp 2 6 12 24 48 96 168

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Sp T2 T6 T12 T24 T48 T96 T168

Mlp-Hesp379-like

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100120140160

Sp 2 6 12 24 48 96 168

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Sp T2 T6 T12 T24 T48 T96 T168

Mlp-Rtp1sc49-like

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Sp 2 6 12 24 48 96 1680

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Sp T2 T6 T12 T24 T48 T96 T168

Mlp-AvrL567-like

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Sp 2 6 12 24 48 96 168

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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.


7- SSP evolutionary constraints

omega ratios

0.0 - 0.2 0.2 - 0.4 0.4 - 0.6 0.6 - 0.8 0.8 - 1.0 1.0 - 1.2 > 1.2

Proportion of HGG's highest pairwise estimates0.0

0.2

0.4

0.6

0.8

Non SecretedSecreted

HGG Loc BLASTX against Fungi nr E-value Cys ω

205 NS No hit ND 2.68

254 NS No hit ND 2.59

1278 NSref|XP_001211022.1| conserved hypothetical protein [Aspergillus terreus]

3e-07 ND 1.88

28 S No hit 0 1.38

92 Sgb|AAS45284.1| proline-rich antigen[Chrysosporium lucknowense]

4e-09 6 1.43

729 Sgb|EAT81533.2| hypothetical protein[Phaeosphaeria nodorum]

7e-11 8 1.32

747 Sref|XP_757360.1| hypothetical protein[Ustilago maydis]

1e-04 4 1.95

4191 S No hit 4 1.57

5606 S No hit 6 1.76

5610 S No hit 6 4.31

5617 S No hit 0 1.23

5624 S No hit 8 1.93

6067 Sref|XP_758577.1| hypothetical protein[Ustilago maydis]

6e-06 10 1.27

Positive selection screen on an EST data set…

Application to whole genome?

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

Acknowledgment

NANCY:Emilie TISSERANTBenoit HILSELBERGERMarie Pierre OUDOT-LE SECQSébastien DUPLESSISFrancis MARTIN

GHENT: Yao-Cheng LINYves van De Peer

JGI sequencing and annotation teamQUEBEC:Nicolas FEAURichard HAMELIN

stéphane hacquard (inra nancy)

Documents

secretome pipeline

pipeline nancy

ssp prediction1669416694

small secreted proteins

voegele et

putative ssp

number of proteins

pipelines nancy