Verteringsenzymen van insecten als doel voor plantaardige afweerstoffen

Proteases from the digestive system of insects are targets of plant defense: the plant produces protease inhibitors to eliminate them. These protease inhibitors are small proteins, which can also be applied in crop protection, for instance through techniques of genetic modification (GMO). However, a number of polyphagous insects, for example Helicoverpa spp., appear not to be affected by protease inhibitors. Being polyphagous, these insects are used to the presence of different natural protease inhibitors in their diet. This may explain why they have adapted and use a protease that cannot be inhibited by a range of protease inhibitors. This protease is homologous to proteases that are extremely sensitive to inhibitors, and we are seeking a molecular understanding of how this adapted protease can avoid being inhibited. Protease inhibitors do work against a number of non-polyphagous insects and also against insects like aphids and thrips, which usually avoid contact with protease inhibitors. Application in agriculture is therefore increasin

Isolation and mapping of a C3'H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3¿-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed map

Antibody orientation on biosensor surfaces: a minireview

Detection elements play a key role in analyte recognition in biosensors. Therefore, detection elements with high analyte specificity and binding strength are required. While antibodies (Abs) have been increasingly used as detection elements in biosensors, a key challenge remains – the immobilization on the biosensor surface. This minireview highlights recent approaches to immobilize and study Abs on surfaces. We first introduce Ab species used as detection elements, and discuss techniques recently used to elucidate Ab orientation by determination of layer thickness or surface topology. Then, several immobilization methods will be presented: non-covalent and covalent surface attachment, yielding oriented or random coupled Abs. Finally, protein modification methods applicable for oriented Ab immobilization are reviewed with an eye to future application

Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions

Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC¿MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formatio

Fruit flavor formation in wild and cultivated strawberry

In recent years we have used various genomics tools to investigate ripening in strawberry, in particular the process of fruit flavor biogenesis. The combination of biochemical analysis, generation of a strawberry Expressed Sequence Tags (EST) collection and gene expression analysis using cDNA microarrays resulted in the identification of several genes playing a key role in the formation of volatile flavors during strawberry fruit ripening. Genes associated with the biosynthesis of lipid-derived flavors, such as esters, and those involved in the formation of mono- and sesquiterpenes have been isolated. In this report we summarize the main results obtained by functional characterization of the genes identified. Moreover, differences in volatile profiles between the wild, diploid strawberry and the cultivated, octaploid strawberry allowed us to obtain insight into the molecular processes which resulted in the formation of a certain flavor component and the loss of anothe

Transcription factor mediated control of anthocyanin biosynthesis in vegetative tissues

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology

Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves

Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases

Caffeoylquinic Acids Biosynthesis and Accumulation in Cynara cardunculus: State of the Art

Plant secondary metabolites are highly evolved compounds performing different functions, and have been widely exploited from food to medicine. A constant supply of phenols, a class of secondary metabolites, provides preventive and defensive mechanisms to reduce the risk of chronic diseases in human beings; among them mono- and di-caffeoylquinic acids (monoCQAs, diCQAs) have attracted a growing academic and industrial interest in recent years. In Cynara cardunculus L. the biosynthetic pathway of chlorogenic acid (CGA, 5-O-caffeoylquinic acid) has been the subject of our several recent studies. Here, we report the state of the art on the isolation and in vitro functional characterization of the genes involved in the biosynthetic pathway of the CGA: HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl-transferase), HQT (hydroxycinnamoyl-CoA quinate hydroxyl-cinnamoyl-transferase), two HQT-like genes, we named Acyltransf_1 and Acyltransf_2, and C3’H (p-coumaroyl ester 3’-hydroxylase). Plant phenolics are known to be involved in the plant stress response and we found out that in globe artichoke the exposure to UV-C induces the production of diCQAs. In UV-C treated globe artichoke leaves, the expression level of C3´H, HCT, HQT, Acyltransf_1, Acyltransf_2 genes was strongly increased, thus confirming their involvement in the synthesis of chlorogenic acid. The development of DNA-based markers for the isolated genes made it possible to locate them within the previously developed genetic maps of the species