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Tomato root exudates from hydroponic cultures

RKN secretome is collected in 3 consecutive stages. Proteins are identified by LC ESI MSMS. Samples. J2 nematode secretions. induced with. Trypsin digested O.N. Tomato root exudate. Secretions. Water. kDa. MW. 1%. 5%. 1%. 5%. 170. Chromatography :

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Tomato root exudates from hydroponic cultures

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  1. RKN secretome is collected in 3 consecutive stages Proteins are identified by LC ESI MSMS Samples J2 nematode secretions induced with Trypsin digested O.N. Tomato root exudate Secretions Water kDa MW 1% 5% 1% 5% 170 Chromatography: 3 phase nanoflow LC-MS/MS reversed phase (RP) and strong cation exchange (SCX) 130 1.5 million J2 stage RKNs Tomato root exudates from hydroponic cultures 95 3500 MW CO 72 4 hours Collect supernatant MS data 2 (0.22 μm filter) 55 Mass spectrometry: Thermo Finnigan LTQ linear ion trap tandem mass spectrometer • Data analysis (Spectrum Mill) • - Refseq -3,239,036 sequences • NCBI nr nematode proteins + A clustered nematode EST database http://www.nematode.net) containing 175,886 sequences • NCBI nr plant protein – 72,265 sequences Electrospray ionization MS data 1 43 4 hours Collectsupernatant J2 stage M. incognita 34 MS data 3 + 0.4 % resorcinol 4-12 % SDS polyacrylamide gel Coloration: Silver staining Whole proteins MS data 4 J2 stage The secretome of M. incognita suggests three main strategies of host-pathogen interaction Specific factors are enriched in the secretome Defense mechanisms 5 4 Translation Gene expression 3 Carbohydrate metabolism and transport Ratio 2 P K Q W R ? C T S A D U I F Y M H 1 V J G N E B O L Z 0 KOGs categories Ratio of secreted proteins vs. Whole proteins (according to their KOGs annotation) Protein turnover/ modification Detoxification Identifying Secretion Proteins of Meloidogyne incognita Patrick Shih, Stephane Bella fiore, Zhouxin Shen Advisor: Steve Briggs Root exudate induces protein secretion from RKN Tomato root exudate and resorcinol induce secretion of partially overlapping protein sets Tomato exudate A. B. 13 5 % Tomato exudate 97.8 % 19 1 Intact J2 79 Intact J2 Resorcinol 16.2 % 34 1,834 246 Resorcinol Nematode database searchreveals 2,226 proteins from secretions plus whole M. incognita at J2 stage. The secretome incudes 392 proteins. Summary The secretome contains at least 711 proteins • Previous studies of plant pathogenic nematodes have identified 10 secreted proteins. All 10 were observed in our dataset of 741 secreted proteins. • Our secretome can be divided into three main categories: • Proteins involved in metabolism and protein degradation (~50%). • Proteins that could be involved in the host-pathogen interaction (~15-20%). • Other • 3. Several proteins could have been acquired by horizontal gene transfer from plants and the question concerning their role in plant-pathogen interaction remains open. NCBI nr Plant database 32 proteins, 72 peptides RefSeqdatabase 335 Nematode database 392 16 2,400 unique peptides revealed 711 proteins (Average of 3.4 peptides per protein)

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