Publications by authors named "Maarten A Jongsma"

53 Publications

Tissue specificity of (E)-β-farnesene and germacrene D accumulation in pyrethrum flowers.

Phytochemistry 2021 Jul 28;187:112768. Epub 2021 Apr 28.

Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China. Electronic address:

Plant defensive mimicry based on the aphid alarm pheromone (E)-β-farnesene (EβF) was previously shown to operate in Tanacetum cinerariifolium (Asteraceae) flowers. Germacrene D (GD), is another dominant volatile of T. cinerariifolium flowers and may modulate both defense and pollination. Here, we find that the increase in GD/EβF ratio at later developmental stages is correlated with the tissue distribution in the flower head: the total content of EβF and GD is similar, but GD accumulates comparatively more in the upper disk florets. Naphthol and N, N-dimethyl-p-phenylenediamine dihydrochloride (NADI)-stained purple ducts containing EβF and GD, were observed in the five petal lips of the corolla and two-lobed stigma of disk florets. By contrast in the peduncle, EβF accounts for nearly 80% of total terpenes, compared to 5% for GD. EβF is accumulated inside inner cortex cells and parenchyma cells of the pith in young peduncle. This is followed by the formation of terpene-filled axial secretory cavities parallel to the vascular bundles. In conclusion, the observed developmental and diurnal emissions of different EβF/GD ratios appear to be regulated by their tissue distribution.
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http://dx.doi.org/10.1016/j.phytochem.2021.112768DOI Listing
July 2021

Development and validation of a UPLC-MS/MS method for the simultaneous determination of gamma-aminobutyric acid and glutamic acid in human plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Feb 28;1164:122519. Epub 2020 Dec 28.

Division of Human Nutrition and Health, Wageningen University & Research, P.O. Box 17, 6700 AA Wageningen, the Netherlands.

Gamma-aminobutyric acid (GABA) and its precursor glutamic acid are important neurotransmitters. Both are also present in peripheral tissues and the circulation, where abnormal plasma concentrations have been linked to specific mental disorders. In addition to endogenous synthesis, GABA and glutamic acid can be obtained from dietary sources. An increasing number of studies suggest beneficial cardio-metabolic effects of GABA intake, and therefore GABA is being marketed as a food supplement. The need for further research into their health effects merits accurate and sensitive methods to analyze GABA and glutamic acid in plasma. To this end, an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of GABA and glutamic acid in human plasma. Samples were prepared by a protein precipitation step and subsequent solid phase extraction using acetonitrile. Chromatographic separation was achieved on an Acquity UPLC HSS reversed phase C18 column using gradient elution. Analytes were detected using electrospray ionization and selective reaction monitoring. Standard curve concentrations for GABA ranged from 3.4 to 2500 ng/mL and for glutamic acid from 30.9 ng/mL to 22,500 ng/mL. Within- and between-day accuracy and precision were <10% in quality control samples at low, medium and high concentrations for both GABA and glutamic acid. GABA and glutamic acid were found to be stable in plasma after freeze-thaw cycles and up to 12 months of storage. The validated method was applied to human plasma from 17 volunteers. The observed concentrations ranged between 11.5 and 20.0 ng/ml and 2269 and 7625 ng/ml for respectively GABA and glutamic acid. The reported method is well suited for the measurement of plasma GABA and glutamic acid in pre-clinical or clinical studies.
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http://dx.doi.org/10.1016/j.jchromb.2020.122519DOI Listing
February 2021

The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay.

Chem Senses 2019 09;44(7):497-505

BU Bioscience, Wageningen University and Research, Droevendaalsesteeg, PB Wageningen, The Netherlands.

The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments.
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http://dx.doi.org/10.1093/chemse/bjz044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357244PMC
September 2019

Defense of pyrethrum flowers: repelling herbivores and recruiting carnivores by producing aphid alarm pheromone.

New Phytol 2019 08 31;223(3):1607-1620. Epub 2019 May 31.

Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.

(E)-β-Farnesene (EβF) is the predominant constituent of the alarm pheromone of most aphid pest species. Moreover, natural enemies of aphids use EβF to locate their aphid prey. Some plant species emit EβF, potentially as a defense against aphids, but field demonstrations are lacking. Here, we present field and laboratory studies of flower defense showing that ladybird beetles are predominantly attracted to young stage-2 pyrethrum flowers that emitted the highest and purest levels of EβF. By contrast, aphids were repelled by EβF emitted by S2 pyrethrum flowers. Although peach aphids can adapt to pyrethrum plants in the laboratory, aphids were not recorded in the field. Pyrethrum's (E)-β-farnesene synthase (EbFS) gene is strongly expressed in inner cortex tissue surrounding the vascular system of the aphid-preferred flower receptacle and peduncle, leading to elongated cells filled with EβF. Aphids that probe these tissues during settlement encounter and ingest plant EβF, as evidenced by the release in honeydew. These EβF concentrations in honeydew induce aphid alarm responses, suggesting an extra layer of this defense. Collectively, our data elucidate a defensive mimicry in pyrethrum flowers: the developmentally regulated and tissue-specific EβF accumulation and emission both prevents attack by aphids and recruits aphid predators as bodyguards.
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http://dx.doi.org/10.1111/nph.15869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6772172PMC
August 2019

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

PLoS One 2019 8;14(4):e0214878. Epub 2019 Apr 8.

Bioscience, Wageningen University & Research, Wageningen, The Netherlands.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214878PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453450PMC
January 2020

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.

New Phytol 2019 07 29;223(2):751-765. Epub 2019 Apr 29.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.
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http://dx.doi.org/10.1111/nph.15821DOI Listing
July 2019

An Integrated System for the Automated Recording and Analysis of Insect Behavior in T-maze Arrays.

Front Plant Sci 2019 29;10:20. Epub 2019 Jan 29.

Wageningen Plant Research, Wageningen University and Research, Wageningen, Netherlands.

Host-plant resistance to insects like thrips and aphids is a complex trait that is difficult to phenotype quickly and reliably. Here, we introduce novel hardware and software to facilitate insect choice assays and automate the acquisition and analysis of movement tracks. The hardware consists of an array of individual T-mazes allowing simultaneous release of up to 90 insect individuals from their individual cage below each T-maze with choice of two leaf disks under a video camera. Insect movement tracks are acquired with computer vision software (EthoVision) and analyzed with EthoAnalysis, a novel software package that allows for automated reporting of highly detailed behavior parameters and statistical analysis. To validate the benefits of the system we contrasted two accessions that were previously analyzed for differential resistance to western flower thrips. Results of two trials with 40 T-mazes are reported and we show how we arrived at optimized settings for the different filters and statistics. The statistics are reported in terms of frequency, duration, distance and speed of behavior events, both as sum totals and event averages, and both for the total trial period and in time bins of 1 h. Also included are higher level analyses with subcategories like short-medium-long events and slow-medium-fast events. The time bins showed how some behavior elements are more descriptive of differences between the genotypes during the first hours, whereas others are constant or become more relevant at the end of an 8 h recording. The three overarching behavior categories, i.e., choice, movement, and halting, were automatically corrected for the percentage of time thrips were detected and 24 out of 38 statistics of behavior parameters differed by a factor 2-6 between the accessions. The analysis resulted in much larger contrasts in behavior traits than reported previously. Compared to leaf damage assays on whole plants or detached leaves that take a week or more to complete, results were obtained in 8 h, with more detail, fewer individuals and higher significance. The potential value of the new integrated system, named EntoLab, for discovery of genetic traits in plants and insects by high throughput screening of large populations is discussed.
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http://dx.doi.org/10.3389/fpls.2019.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361829PMC
January 2019

Automated high-throughput individual tracking system for insect behavior: Applications on memory retention in parasitic wasps.

J Neurosci Methods 2018 11 15;309:208-217. Epub 2018 Sep 15.

Laboratory of Entomology, Plant Sciences Group, Wageningen University, Wageningen, the Netherlands.

Background: Insects are important models to study learning and memory formation in both an ecological and neuroscience context due to their small size, behavioral flexibility and ecological diversity. Measuring memory retention is often done through simple time-consuming set-ups, producing only a single parameter for conditioned behavior. We wished to obtain higher sample sizes with fewer individuals to measure olfactory memory retention more efficiently.

New Method: The high-throughput individual T-maze uses commercially available tracking software, Ethovision XT, in combination with a Perspex stack of plates as small as 18 × 18 cm, which accommodates 36 olfactory T-mazes, where each individual wasp could choose between two artificial odors. Various behavioral parameters, relevant to memory retention, were acquired in this set-up; first choice, residence time, giving up time and zone entries. From these parameters a performance index was calculated as a measure of memory retention. Groups of 36 wasps were simultaneously tested within minutes, resulting in efficient acquisition of sufficiently high sample sizes.

Results: This system was tested with two very different parasitic wasp species, the larval parasitoid Cotesia glomerata and the pupal parasitoid Nasonia vitripennis, and has proven to be highly suitable for testing memory retention in both these species.

Comparison With Existing Methods: Unlike other bioassays, this system allows for both high-throughput and recording of detailed individual behavior.

Conclusions: The high-throughput individual T-maze provides us with a standardized high-throughput, labor-efficient and cost-effective method to test various kinds of behavior, offering excellent opportunities for comparative studies of various aspects of insect behavior.
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http://dx.doi.org/10.1016/j.jneumeth.2018.09.012DOI Listing
November 2018

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Sensors (Basel) 2018 Feb 16;18(2). Epub 2018 Feb 16.

BU Bioscience, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.
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http://dx.doi.org/10.3390/s18020602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855233PMC
February 2018

Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids.

Plant Biotechnol J 2018 08 9;16(8):1434-1445. Epub 2018 Feb 9.

Key Laboratory of Horticultural Plant Biology, MOE Huazhong Agricultural University, Wuhan, China.

Aphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and nonvolatile metabolite profiles can disrupt aphid-plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over-expressed in Chrysanthemum morifolium and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl-6-O-malonyl-β-D-glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual-choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open-field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defence system, with both repellence by chrysanthemol odour and deterrence by its nonvolatile glycoside, introducing a promising new option for engineering aphid control into plants.
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http://dx.doi.org/10.1111/pbi.12885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041446PMC
August 2018

SIEVE ELEMENT-LINING CHAPERONE1 Restricts Aphid Feeding on Arabidopsis during Heat Stress.

Plant Cell 2017 Oct 28;29(10):2450-2464. Epub 2017 Sep 28.

Bioscience, Wageningen University & Research, 6708 PB Wageningen, The Netherlands.

The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural accessions, we identified the small heat shock-like (). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when expression is upregulated. At this moderately high temperature, mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress.
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http://dx.doi.org/10.1105/tpc.16.00424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774557PMC
October 2017

Genetic architecture of plant stress resistance: multi-trait genome-wide association mapping.

New Phytol 2017 Feb 4;213(3):1346-1362. Epub 2016 Oct 4.

Wageningen University and Research Plant Breeding, Wageningen University and Research, PO Box 386, 6700 AJ, Wageningen, the Netherlands.

Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker-trait associations in genome-wide association (GWA) analyses using tailored multi-trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad-spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below-ground vs above-ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.
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http://dx.doi.org/10.1111/nph.14220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5248600PMC
February 2017

AtWRKY22 promotes susceptibility to aphids and modulates salicylic acid and jasmonic acid signalling.

J Exp Bot 2016 05 23;67(11):3383-96. Epub 2016 Apr 23.

Plant Research International, Business Unit Bioscience, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, The Netherlands.

Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA-SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences.
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http://dx.doi.org/10.1093/jxb/erw159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892728PMC
May 2016

Automated video tracking of thrips behavior to assess host-plant resistance in multiple parallel two-choice setups.

Plant Methods 2016 18;12. Epub 2016 Jan 18.

PRI-Bioscience, Wageningen University and Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands.

Background: Piercing-sucking insects cause severe damage in crops. Breeding for host-plant resistance can significantly reduce the yield losses caused by these insects, but host-plant resistance is a complex trait that is difficult to phenotype quickly and reliably. Current phenotyping methods mainly focus on labor-intensive and time-consuming end-point measurements of plant fitness. Characterizing insect behavior as a proxy for host-plant resistance could be a promising time-saving alternative to end-point measurements.

Results: We present a phenotyping platform that allows screening for host-plant resistance against Western flower thrips (WFT, Frankliniella occidentalis (Pergande)) in a parallel two-choice setup using automated video tracking of thrips behavior. The platform was used to establish host-plant preference of WFT with a large plant population of 345 wild Arabidopsis accessions and the method was optimized with two extreme accessions from this population that differed in resistance towards WFT. To this end, the behavior of 88 WFT individuals was simultaneously tracked in 88 parallel two-choice arenas during 8 h. Host-plant preference of WFT was established both by the time thrips spent on either accession and various behavioral parameters related to movement (searching) and non-movement (feeding) events.

Conclusion: In comparison to 6-day end-point choice assays with whole plants or detached leaves, the automated video-tracking choice assay developed here delivered similar results, but with higher time- and resource efficiency. This method can therefore be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations.
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http://dx.doi.org/10.1186/s13007-016-0102-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717623PMC
January 2016

High-throughput phenotyping of plant resistance to aphids by automated video tracking.

Plant Methods 2015 30;11. Epub 2015 Jan 30.

Plant Research International, Wageningen University and Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands.

Background: Piercing-sucking insects are major vectors of plant viruses causing significant yield losses in crops. Functional genomics of plant resistance to these insects would greatly benefit from the availability of high-throughput, quantitative phenotyping methods.

Results: We have developed an automated video tracking platform that quantifies aphid feeding behaviour on leaf discs to assess the level of plant resistance. Through the analysis of aphid movement, the start and duration of plant penetrations by aphids were estimated. As a case study, video tracking confirmed the near-complete resistance of lettuce cultivar 'Corbana' against Nasonovia ribisnigri (Mosely), biotype Nr:0, and revealed quantitative resistance in Arabidopsis accession Co-2 against Myzus persicae (Sulzer). The video tracking platform was benchmarked against Electrical Penetration Graph (EPG) recordings and aphid population development assays. The use of leaf discs instead of intact plants reduced the intensity of the resistance effect in video tracking, but sufficiently replicated experiments resulted in similar conclusions as EPG recordings and aphid population assays. One video tracking platform could screen 100 samples in parallel.

Conclusions: Automated video tracking can be used to screen large plant populations for resistance to aphids and other piercing-sucking insects.
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http://dx.doi.org/10.1186/s13007-015-0044-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318543PMC
February 2015

Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

J Biol Chem 2014 Dec 5;289(52):36325-35. Epub 2014 Nov 5.

From Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands,

Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.
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http://dx.doi.org/10.1074/jbc.M114.623348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276892PMC
December 2014

Development of late blight resistant potatoes by cisgene stacking.

BMC Biotechnol 2014 May 29;14:50. Epub 2014 May 29.

Wageningen UR Plant Breeding, Wageningen University & Research Centre, P,O, Box 386, 6700 AJ Wageningen, The Netherlands.

Background: Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.

Results: We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.

Conclusions: We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
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http://dx.doi.org/10.1186/1472-6750-14-50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075930PMC
May 2014

Orientation of llama antibodies strongly increases sensitivity of biosensors.

Biosens Bioelectron 2014 Oct 18;60:130-6. Epub 2014 Apr 18.

Plant Research International, Wageningen, The Netherlands. Electronic address:

Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics.
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http://dx.doi.org/10.1016/j.bios.2014.04.017DOI Listing
October 2014

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Metab Eng 2013 Nov 21;20:198-211. Epub 2013 Sep 21.

Laboratory of Plant Physiology, Wageningen UR, P.O. Box 658, 6700 AR Wageningen, The Netherlands.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.
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http://dx.doi.org/10.1016/j.ymben.2013.09.002DOI Listing
November 2013

A trichome-specific linoleate lipoxygenase expressed during pyrethrin biosynthesis in pyrethrum.

Lipids 2013 Oct 28;48(10):1005-15. Epub 2013 Jul 28.

Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.

The lipid precursor alcohols of pyrethrins-jasmolone, pyrethrolone and cinerolone-have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases, TcLOX1 and TcLOX2, that have a similar pattern to TcCDS. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as TcCDS, TcLOX1, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.
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http://dx.doi.org/10.1007/s11745-013-3815-1DOI Listing
October 2013

Comparative antifeedant activities of polygodial and pyrethrins against whiteflies (Bemisia tabaci) and aphids (Myzus persicae).

Pest Manag Sci 2014 Apr 12;70(4):682-8. Epub 2013 Aug 12.

Plant Research International, Wageningen, The Netherlands; Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.

Background: Polygodial, a sesquiterpene dialdehyde of the drimane family, has been shown to have deterrent and antifeedant effects on various insect species, including Myzus persicae (Sulzer), Spodoptera spp. and Leptinotarsa decemlineata (Say). This compound may have potential as a broad-spectrum biocontrol agent, similar to pyrethrins, given that it was previously reported to improve yield when sprayed on barley fields.

Results: This study compares the deterrent effect of polygodial and pyrethrins against the silverleaf whitefly Bemisia tabaci (Gennadius) and the green peach aphid M. persicae in dual-choice assays using compound-coated tomato leaf discs. B. tabaci adults were deterred by polygodial at an ED50 (effective dose at which 50% of the insects are deterred) of about 25 µg g(-1) fresh weight (FW), and green peach aphids at about 54 µg g(-1) FW. Bioassays were benchmarked with pyrethrins that had a 20-fold lower ED50 of approximately 1.4 µg g(-1) FW against whiteflies, but only a twofold lower ED50 (about 28 µg g(-1) FW) against peach aphids. Polygodial showed moderate phytotoxic effects (score of 2 on a scale of 1-5) on tomato leaves at concentrations above the ED50 concentrations (≥ 90 µg g(-1) FW).

Conclusion: The sesquiterpene dialdehyde polygodial is 2-20 times less deterrent than pyrethrins, depending on the insect species, but it could provide a useful complement to pyrethrin sprays as it has a different mode of action, is food grade and has low volatility. However, a formulation that reduces the risks of phytotoxic effects should be developed.
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http://dx.doi.org/10.1002/ps.3610DOI Listing
April 2014

Chrysanthemum expressing a linalool synthase gene 'smells good', but 'tastes bad' to western flower thrips.

Plant Biotechnol J 2013 Sep 7;11(7):875-82. Epub 2013 Jun 7.

Plant Research International, Wageningen UR, Wageningen, The Netherlands; Laboratory of Entomology, Wageningen UR, Wageningen, The Netherlands.

Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound.
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http://dx.doi.org/10.1111/pbi.12080DOI Listing
September 2013

Biosynthesis of sesquiterpene lactones in pyrethrum (Tanacetum cinerariifolium).

PLoS One 2013 31;8(5):e65030. Epub 2013 May 31.

Plant Research International, Wageningen University and Research Centre, Wageningen, The Netherlands.

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or β) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3669400PMC
January 2014

16 kDa heat shock protein from heat-inactivated Mycobacterium tuberculosis is a homodimer - suitability for diagnostic applications with specific llama VHH monoclonals.

PLoS One 2013 30;8(5):e64040. Epub 2013 May 30.

Plant Research International, Wageningen, The Netherlands.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064040PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667823PMC
January 2014

A generic microfluidic biosensor of G protein-coupled receptor activation-monitoring cytoplasmic [Ca(2+)] changes in human HEK293 cells.

Biosens Bioelectron 2013 Sep 3;47:436-44. Epub 2013 Apr 3.

Plant Research International, Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca(2+)] rises that return to basal levels in 30-60s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca(2+)] changes in real-time using Förster (Fluorescence) resonance energy transfer (FRET). On this basis, we designed the prototype of a generic microfluidic biosensor of GPCR activation, imaging [Ca(2+)] changes in recombinant human HEK293 cells, which express a combination of a GPCR (Neurokinin 1) and YC3.6. An internal reference for non-specifically induced [Ca(2+)] changes were YC3.6 cells without GPCR but expressing a red fluorescent protein (mCherry) for identification. These cell lines were grown as a mixed population in a flow cell with a volume of ~50µl and a flow cell surface of 170mm(2). Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5-100µl) at a flow speed of 100µl/min. A flow cell surface of 0.2mm(2) with 50 cells was imaged every 2-4s to obtain signal kinetics. The lower limit of detection was 30pM Substance P (SP, 2pg/50µl), and reproducible responses to repeated injections every 3min were obtained at 1nM SP. This biosensor was designed for ~50 cells for statistical reasons, but at a lower limit of 1 receptor- and 1 reference-cell, specific ligand detection is still feasible.
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http://dx.doi.org/10.1016/j.bios.2013.03.065DOI Listing
September 2013

Bidirectional secretions from glandular trichomes of pyrethrum enable immunization of seedlings.

Plant Cell 2012 Oct 26;24(10):4252-65. Epub 2012 Oct 26.

Plant Research International, Wageningen University and Research Centre, 6708 PB Wageningen, The Netherlands.

Glandular trichomes are currently known only to store mono- and sesquiterpene compounds in the subcuticular cavity just above the apical cells of trichomes or emit them into the headspace. We demonstrate that basipetal secretions can also occur, by addressing the organization of the biosynthesis and storage of pyrethrins in pyrethrum (Tanacetum cinerariifolium) flowers. Pyrethrum produces a diverse array of pyrethrins and sesquiterpene lactones for plant defense. The highest concentrations accumulate in the flower achenes, which are densely covered by glandular trichomes. The trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. However, during achene maturation, the key pyrethrin biosynthetic pathway enzyme chrysanthemyl diphosphate synthase is expressed only in glandular trichomes. We show evidence that chrysanthemic acid is translocated from trichomes to pericarp, where it is esterified into pyrethrins that accumulate in intercellular spaces. During seed maturation, pyrethrins are then absorbed by the embryo, and during seed germination, the embryo-stored pyrethrins are recruited by seedling tissues, which, for lack of trichomes, cannot produce pyrethrins themselves. The findings demonstrate that plant glandular trichomes can selectively secrete in a basipetal direction monoterpenoids, which can reach distant tissues, participate in chemical conversions, and immunize seedlings against insects and fungi.
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http://dx.doi.org/10.1105/tpc.112.105031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517248PMC
October 2012

Pyrethrins protect pyrethrum leaves against attack by western flower thrips, Frankliniella occidentalis.

J Chem Ecol 2012 Apr 29;38(4):370-7. Epub 2012 Mar 29.

Plant Research International, Wageningen UR, P.O. Box 619, 6700 AP, Wageningen, The Netherlands.

Pyrethrins are active ingredients extracted from pyrethrum flowers (Tanacetum cinerariifolium), and are the most widely used botanical insecticide. However, several thrips species are commonly found on pyrethrum flowers in the field, and are the dominant insects found inside the flowers. Up to 80% of western flower thrips (WFT, Frankliniella occidentalis) adults died within 3 days of initiating feeding on leaves of pyrethrum, leading us to evaluate the role of pyrethrins in the defense of pyrethrum leaves against WFT. The effects of pyrethrins on WFT survival, feeding behavior, and reproduction were measured both in vitro and in planta (infiltrated leaves). The lethal concentration value (LC50) for pyrethrins against WFT adults was 12.9 mg/ml, and pyrethrins at 0.1% (w/v) and 1% (w/v) had significantly negative effects on feeding, embryo development, and oviposition. About 20-70% of WFT were killed within 2 days when they were fed chrysanthemum leaves containing 0.01-1% pyrethrins. Chrysanthemum leaves containing 0.1% or 1% pyrethrins were significantly deterrent to WFT. In a no-choice assay, the reproduction of WFT was reduced significantly when the insects were fed leaves containing 0.1% pyrethrins, and no eggs were found in leaves containing 1% pyrethrins. Our results suggest that the natural concentrations of pyrethrins in the leaves may be responsible for the observed high mortality of WFT on pyrethrum.
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http://dx.doi.org/10.1007/s10886-012-0097-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324680PMC
April 2012

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense.

Arch Insect Biochem Physiol 2012 Mar;79(3):153-81

Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.
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http://dx.doi.org/10.1002/arch.21017DOI Listing
March 2012

Association mapping of plant resistance to insects.

Trends Plant Sci 2012 May 8;17(5):311-9. Epub 2012 Feb 8.

Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH Wageningen, The Netherlands.

Association mapping is rapidly becoming an important method to explore the genetic architecture of complex traits in plants and offers unique opportunities for studying resistance to insect herbivores. Recent studies indicate that there is a trade-off between resistance against generalist and specialist insects. Most studies, however, use a targeted approach that will easily miss important components of insect resistance. Genome-wide association mapping provides a comprehensive approach to explore the whole array of plant defense mechanisms in the context of the generalist-specialist paradigm. As association mapping involves the screening of large numbers of plant lines, specific and accurate high-throughput phenotyping (HTP) methods are needed. Here, we discuss the prospects of association mapping for insect resistance and HTP requirements.
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http://dx.doi.org/10.1016/j.tplants.2012.01.002DOI Listing
May 2012

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

PLoS One 2011 26;6(10):e26754. Epub 2011 Oct 26.

Plant Research International, Wageningen, The Netherlands.

Background: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.

Methodology/principal Findings: Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4 × 10(-10) M.

Conclusions/significance: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026754PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202562PMC
March 2012