Mycoparasitism related targets of Tmk1 indicate stimulating regulatory functions of this MAP kinase in Trichoderma atroviride.

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Validation and comparison of a sandwich ELISA, two competitive ELISAs and a real-time PCR method for the detection of lupine in food.

Methods applied in food allergen analysis should be specific, sensitive and applicable to both raw and highly processed foods. The performance of the most commonly used methods, ELISA and real-time PCR, may, however, be influenced by food processing steps, e.g., heat treatment. The present study compares the applicability of four in-house developed methods, one sandwich ELISA, two competitive ELISAs and a real-time PCR method, for the detection of lupine in four different food matrices, comprising bread, biscuits, rice patties and noodles. In order to investigate the influence of food processing on the detectability, not only the heat treated model foods but also the corresponding doughs were analysed. The sandwich ELISA proved to be the most sensitive method. The LOD was found to be 10 ppm lupine, independent from the food matrix and independent if the dough or the heat treated food was analysed. In addition, the methods were applied to the analysis of commercial foodstuffs differing in their labelling.

Development and validation of a sandwich ELISA for the determination of potentially allergenic sesame (Sesamum indicum) in food.

This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows the determination of traces of sesame in food. Chicken anti-sesame antibodies, used as coating antibodies, and rabbit anti-sesame antibodies, used as secondary antibodies, were prepared by immunization with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 19 food ingredients commonly found in sesame-containing foodstuffs such as seeds, nuts, and cereals. In whole grain bread, crisp toast, and snacks, the limit of detection (S/N = 3) was 0.5, 0.5, and 0.3 µg sesame protein/g, and the limit of quantification (S/N = 10) was 0.6, 0.8, and 1.4 µg sesame protein/g, respectively. The analysis of blank food matrices (whole grain bread, white bread, crisp toast, and snacks) spiked with sesame protein at four spike levels generally resulted in mean recoveries from 72% to 145%. In the case of spiking blank food matrices with sesame seeds, the ELISA proved to be more accurate for whole wheat cookies than for whole wheat bread.

Development and validation of an indirect competitive enzyme linked-immunosorbent assay for the determination of potentially allergenic sesame (Sesamum indicum) in food.

This study was designed to develop an indirect competitive enzyme linked-immunosorbent assay (ELISA) to detect traces of sesame in food. Antibodies against sesame were prepared by immunizing a hen with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 12 of 13 food ingredients tested, only for chocolate was a low cross-reactivity of 0.7% observed. To eliminate matrix effects, sesame protein standard solutions were prepared by diluting the sesame extract with blank food matrix (1:20 diluted with PBS). Recovery of sesame protein in food samples (crisp toasts, snacks, and rolls) spiked with different sesame protein concentrations ranged from 85% to 120%, with the exception of multigrain crisp toast, resulting in too high recoveries (117%-160%) and whole grain bread, yielding too low recoveries (70%-85%). In crisp bread, cracker, cereals, and snacks the limit of detection (LOD) was found to be 5 microg of sesame protein/g of food, in fresh breads and rolls, the LOD was 11 microg of sesame protein/g of food.