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.