Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of P12-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by P12, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by P12. P12-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.The involvement of endogenous proteinaceous proteinase inhibitors (PIs) in plant defense against leaf-feeding insects has been well recognized (1, 2). Experiments with artificial diets and a wide range of insects confirmed the antinutritional effects of proteinaceous PIs (3-7), although some negative results were also reported (8). The expression of heterologous PIs in transgenic tobacco plants provided final confirmation in planta for their roles as resistance factors, although protection was only partial (9, 10).Recently, the effectiveness of proteinase inhibitors was suggested to depend on the affinity or specificity of an inhibitor for the main gut proteinases of an insect (4,11,12), but the mechanism of action and effect of proteinase inhibitors are only partially understood. Broadway and Duffey (3) showed that gut proteinase activities of Spodoptera exigua and Heliothis zea were similar or increased when larvae were chronically exposed to high levels of potato proteinase inhibitor II (PI2) or soybean trypsin inhibitor in artificial diets. The simple scenario that growth rates were reduced due to reduced rates of proteolysis (13) was, therefore, dismissed. Instead, these results were interpreted by Broadway and Duffey (3) to suggest that a feedback mechanism was leading to the hyperproduction of proteinases to compensate for the loss of activity, which in turn led to the depletion of essential amino acids and finally resulted in retarded growth rates.Our aim was to establish to what extent and how PI2 expressed in tobacco leaves would affect larval growth and digestive physiology of S. exigua. Our data show that S. exigua larvae adapt to PIs by induction of gut proteinase activity that is insensitive to inhibition.t MATERIALS AND METHODSPlant Transformation. A full-length PI2 cDNA clone, p303.5 1, was selected from a tuber-specific Solanum tuberosum cv. Bintje cDNA library by using p303 as a probe (14, 15). The region encoding the N-terminal part was amplified by PCR using primers 152FO (5'-GCGGGATCCACCATGGC...

A 6-plex competitive inhibition immunoassay for mycotoxins in barley was developed on a prototype portable nanostructured imaging surface plasmon resonance (iSPR) instrument, also referred to as imaging nanoplasmonics. As a benchmark for the prototype nanoplasmonics instrument, first a double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using a well-established benchtop SPR instrument and two biosensor chips. To this end, ovalbumin (OVA) conjugates of mycotoxins were immobilized on the chip via amine coupling. The SPR response was then recorded upon injection of a mixture of antibodies at a fixed concentration and the sample (or matrix-matched standard) over a chip with immobilized mycotoxin-OVA conjugates. The chips were regenerated after each sample using 10 mM HCl and 20 mM NaOH and could be used for at least 60 cycles. The limits of detection in barley (in μg kg(-1)) were determined to be 26 for DON, 6 for ZEA, 0.6 for T-2, 3 for OTA, 2 for FB1 and 0.6 for AFB1. Preliminary in-house validation showed that DON, T-2, ZEA and FB1 can be detected at the European Union regulatory limits, while for OTA and AFB1 sensitivities should be improved. Furthermore, measurement of naturally contaminated barley showed that the assay can be used as a semi-quantitative screening method for mycotoxins prior to liquid chromatography tandem mass spectrometry (LC-MS/MS). Finally, using the same bio-reagents the assay was transferred to a 6-plex format in the nanoplasmonics instrument and subsequently the two assays were compared. Although less sensitive, the 6-plex portable iSPR assay still allowed detection of DON, T-2, ZEA and FB1 at relevant levels. Therefore, the prototype iSPR shows potential for future applications in rapid in-field and at-line screening of multiple mycotoxins.

Currently beer is booming, mainly due to the steady rise of craft breweries worldwide. Previous surveys for occurrence of mycotoxins in beer, were mainly focussed on industrial produced beer. The present survey reports the presence of mycotoxins in craft beer and how this compares to industrial produced beer. More than 1000 beers were collected from 47 countries, of which 60% were craft beers. A selection of 1000 samples were screened for the presence of aflatoxin B1, ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FBs), T-2 and HT-2 toxins (T-2 and HT-2) and deoxynivalenol (DON) using a mycotoxin 6-plex immunoassay. For confirmatory analysis, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and applied. The 6-plex screening showed discrepancies with the LC-MS/MS analysis, possibly due to matrix interference and/or the presence of unknown mycotoxin metabolites. The major mycotoxins detected were DON and its plant metabolite deoxynivalenol-3-β-D-glucopyranoside (D3G). The 6-plex immunoassay reported the sum of DON and D3G (DON+D3G) contaminations ranging from 10 to 475 μg/L in 406 beers, of which 73% were craft beers. The popular craft beer style imperial stout, had the highest percentage of samples suspected positive (83%) with 29% of all imperial stout beers having DON+D3G contaminations above 100 μg/L. LC-MS/MS analysis showed that industrial pale lagers from Italy and Spain, predominantly contained FBs (3–69 μg/L). Besides FBs, African traditional beers also contained aflatoxins (0.1–1.2 μg/L). The presence of OTA, T-2, HT-2, ZEN, β-zearalenol, 3/15-acetyl-DON, nivalenol and the conjugated mycotoxin zearalenone 14-sulfate were confirmed in some beers. This study shows that in 27 craft beers, DON+D3G concentrations occurred above (or at) the Tolerable Daily Intake (TDI). Exceeding the TDI, may have a health impact. A better control of brewing malts for craft beer, should be put in place to circumvent this potential problem.

A multi-mycotoxin immunoassay—using the MultiAnalyte Profiling (xMAP) technology—is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability.

Transgenic plants are increasingly used as production platforms for various proteins, yet protein expression levels in the range of the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase have not yet been achieved by nuclear transformation. Suitable gene regulatory 5¢ and 3¢ elements are crucial to obtain adequate expression. In this study an abundantly transcribed member (rbcS1) of the ribulose-1,5-bisphosphate carboxylase small-subunit gene family of chrysanthemum (Chrysanthemum morifolium Ramat.) was cloned. The promoter of rbcS1 was found to be homologous to promoters of highly expressed rbcS gene members of the plant families Asteraceae, Fabaceae and Solanaceae. The regulatory 5¢ and 3¢ non-translated regions of rbcS1 were engineered to drive heterologous expression of various genes. In chrysanthemum, the homologous rbcS1 cassette resulted in a b-glucuronidase (gusA) accumulation of, at maximum, 0.88% of total soluble protein (population mean 0.17%). In tobacco (Nicotiana tabacum L.), the gusA expression reached 10% of total soluble protein. The population mean of 2.7% was found to be 7-to 8-fold higher than for the commonly used cauliflower mosaic virus (CaMV) 35S promoter (population mean 0.34%). RbcS1-driven expression of sea anemone equistatin in potato (Solanum tuberosum L.), and potato cystatin in tomato (Lycopersicon esculentum Mill.) yielded maximum levels of 3-7% of total soluble protein. The results demonstrate, that the compact 2-kb rbcS1 expression cassette provides a novel nuclear transformation vector that generates plants with expression levels of up to 10% of total protein.