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View Ecp6 Proteins

Ecp6 (extracellular protein 6) is a effector protein (virulence factor) of Cladosporium fulvum that prevents trigger host immunity of tomato (Solanum lycopersicum), when cell wall chitin oligosaccharide of the microorganism are released to the symplast. Ecp6 contains three LysM domains that binding chitin. On tomato cells, it has been proved that inhibit chitin-induced medium alkalization and ROS (reactive oxygen species) production, a response related with plant resistance and exoquitinase action1. Ecp6 is homolog to Colletotrichum lindemuthianum protein CIH1 of the intracellular hyphae2.

LysM (Lysin motif) is a carbohydrate-binding motif present on different proteins. LysM is present on prokaryotes and plants (like a surface receptor to nodulation process): Rhizobium- legumes symbiosis, Arabidopsis, rice (Oryza sativa). On fungal, approximately 4000 LysM proteins have been identified2.

Results

Some comparisons were done between coffee rust (Hemileia vastatrix) contigs and Cladosporium fulvum Ecp6 protein. The experiments and their results are summarized bellow:

1. BlastX, Blosum and E-value = 1E-3.

CONTIG IDENTITY COVERAGE E-VALUE
contig01156 51 15 3e-04
contig01385 30 16 0,001
contig08063 30 7 0,001
contig09375 43 9 4e-04
contig12896 39 8 0,001
contig19667 28 14 0,001
contig22533 29 22 0,001
contig27715 35 11 5e-04
contig37554 76 7 0,001
contig41166 47 40 0,001

2. BlastP with Augustus protein predictions of rust DNA contigs versus Ecp6, PAM30 1E-1.

Model Augusuts IDENTITY COVERAGE E-VALUE CONTIG
Saccharomyces_58 83,33 6 0,054 contig00951
Saccharomyces_1835 34,48 29 0,006 contig22519
Saccharomyces_2057 60,00 10 0,079 contig25770
Saccharomyces_2219 75,00 8 0,076 contig29342

3. PSI-blast (through blastpgp) with Augustus protein predictions of rust DNA contigs versus Ecp6, E-value = 1E-2, J-10 (number of iterations). Results are shown for each contig and iteration:

Mode IDENTITY COVERAGE E-VALUE CONTIG
Saccharomyces_264 26.53 49 0.006 contig03107
Saccharomyces_264 26.53 49 9e-04
Saccharomyces_264 26.53 49 4e-23
Saccharomyces_264 21.43 28 2e-04
Saccharomyces_317 44.00 25 6e-04 contig03707
Saccharomyces_317 44.00 25 3e-11
Saccharomyces_856 42.86 21 0.001 contig09578
Saccharomyces_856 42.86 21 3e-10
Saccharomyces_856 18.18 22 0.003
Saccharomyces_1420 36.00 25 0.002 contig16306
Saccharomyces_1420 36.00 25 3e-13
Saccharomyces_1530 23.68 38 0.004 contig17778
Saccharomyces_1530 30.43 23 0.008
Saccharomyces_1748 50.00 16 0.004 contig21148
Saccharomyces_1748 50.00 16 0.005
Saccharomyces_1835 34.62 26 0.002 contig22519
Saccharomyces_1835 34.48 29 0.001
Saccharomyces_1835 34.48 29 5e-14
Saccharomyces_2459 31.48 54 0.002 contig33951
Saccharomyces_2459 26.83 82 1e-25

4. Also, matched proteins by PSI-Blast on step 3 were ran on web-based HMMER vs Pfam database (pfam_scan.pl) to annoted motif on these. Searching against: /data/blastdb/Pfam/data_24/Pfam-A.hmm, with options: cut off -E 1.0 —domE 1.0, resolve clan overlaps: on, predict active sites: on. Results are shown below:

Model HMM_Acc HMM_Name Type HMM_Length Bit_score E-value Signif Clan Contig
Saccharomyces_317 PF02194.8 PXA Family 184 67.9 6.3e-19 1 No_clan contig03707
Saccharomyces_1420 PF11488.1 Lge1 Family 274 11.8 0.11 0 No_clan contig16306
Saccharomyces_1530 PF07177.5 Neuralized Family 69 11.6 0.15 0 No_clan contig17778
Saccharomyces_1748 PF00665.19 rve Domain 120 13.5 0.045 0 CL021 contig21148

5. Finally, protein predictions of Augustus on coffee rust were compared with LysM HMM profile of Pfam (PF01476). For this, HMMER tools were used (v. 3.0)3. No proteins were found that matched LysM motif.

References

1. Ronnie de Jonge, H. Peter van Esse, Anja Kombrink, Tomonori Shinya, Yoshitake Desaki, Ralph Bours, Sander van der Krol, Naoto Shibuya, Matthieu H. A. J. Joosten, Bart P. H. J. Thomma. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329, 953, 2010.

2. Ronnie de Jonge and Bart P.H.J. Thomma. Fungal LysM effectors: extinguishers of host immunity? Trends. Microb. 2009.

3. The theory behind profile HMMs: R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998.