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The secretome of Melampsora larici-populina First
results
Stéphane HACQUARD (INRA NANCY)
David JOLY (CFL QUEBEC)
Nancy, workshop Melampsora, august 2008
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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)
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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
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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
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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
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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
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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
Gene models ? 300 amino acids
LENGTH
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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)
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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
Melampsora Puccinia Laccaria
Gene models 16694 20567 20613
SSP ? 300aa 985 (5,8) 1219 (5,9) 864 (4,2)
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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
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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 (largest14 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 gt S or S gt
NS)
? 601 SSP correspond to single gene model
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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 gt S
? Among 155 SSP manually curated corresponding to
single model 45 show homology with others gene
models (blastp) or genomic regions (tblastn) gt
New families
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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
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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
Homology with HESP-417
Hypothetical protein
Hypothetical protein
Hypothetical protein
Scaffold 31
Cutinase
Homology with RTP1
Cutinase
1kb
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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
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III- The secretome of Melampsora larici-populina
6- SSP expression data
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
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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
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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
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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.

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III- The secretome of Melampsora larici-populina
7- SSP evolutionary constraints
Positive selection screen on an EST data set
? Application to whole genome?
HGG Loc BLASTX against Fungi nr E-value Cys ?
205 NS No hit ND 2.68
254 NS No hit ND 2.59
1278 NS refXP_001211022.1 conserved hypothetical protein Aspergillus terreus 3e-07 ND 1.88
28 S No hit 0 1.38
92 S gbAAS45284.1 proline-rich antigen Chrysosporium lucknowense 4e-09 6 1.43
729 S gbEAT81533.2 hypothetical protein Phaeosphaeria nodorum 7e-11 8 1.32
747 S refXP_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 S refXP_758577.1 hypothetical protein Ustilago maydis 6e-06 10 1.27
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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
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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 team
QUEBEC Nicolas FEAU Richard HAMELIN