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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.

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. (C) 2012 Wiley Periodicals, Inc.

In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multidomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM)] were fused into reading frames consisting of four (K-A-C-P) to five (EIM-K-A-C-P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multidomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14-16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20?f the control. Leaf damage on protected plants was minimal. Engineered multidomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.

Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nm to 30 nm. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.

Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66 f the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76 dentical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nm to 30 nm. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.

To attain high transgene expression in petal tissue of ray florets of chrysanthemum an endogenous ubiquitin extension protein (UEP1) promoter was cloned and tested with the β-glucuronidase (GUS) reporter gene. Expression levels were compared with four heterologous promoters: chalcone synthase (chs-A) and zinc finger transcription factor (EPF2-5) from petunia, eceriferum (CER6) from Arabidopsis and multicystatin (PMC) from potato. The comparison of the expression levels of the different constructs in ray florets, disc florets, and leaves is presented. The highest mean expression in petal tissue of ray and disc florets was conferred by the UEP1 promoter, followed by CER6 and EPF2-5. The UEP1 promoter in ray florets confers over 50-fold enhancement in expression as compared to CaMV 35S-based promoters.

Transgenic Spodoptera exigua are being developed by microinjection of a piggyBac vector. The vector expresses green fluorescent protein (GFP) under the control of the actin promoter. Forty percent of the first-instar larvae that hatched from the injected eggs were green fluorescent. However, after backcrossing none of the G1 first-instar larvae was fluorescent and a transgenic line could not be established. Several possibilities for the use of transgenic insects are discussed

Plants and herbivores have been co-evolving for thousands of years, and as a result, plants have defence mechanisms that offer protection against many herbivores such as nematodes, insects, birds and mammals. Only when a herbivore has managed to adapt to these defence mechanisms does it have the potential to become a pest. One such method of plant defence involves the production of protease inhibitors (PIs). These inhibitors are proteins that may be found constitutively in various parts of the plant, or may be induced in response to herbivore attack. PIs work at the gut level, by inhibiting the digestion of plant protein. This review focuses on insect herbivores and looks at the mechanisms involved in the role and function of PIs in plant defense against insects, as well as at the ability of well adapted species to overcome the effects of these plant PIs.

The research reported in this thesis describes the induction of proteinase inhibitor synthesis in solanaceous plants (tobacco and tomato), when lepidopteran larvae (Manduca sexta and Spodoptera exigua) are feeding on leaves. It is shown that the larvae circumvent the proteinase inhibitor defense of these plants by the induction of non-susceptible gut proteinases. A phage display method is presented, which may allow the isolation of PIs that are also active against the non-susceptible proteinases of insects. It is expected that the application of such PIs can complement the natural PI defense of plants, and result in the protection of transgenic plants against insects.Chapter one provides a general introduction to plant PIs It describes the different PI families identified in plants, their mode of action against serine proteinases and the available evidence for a defensive role in plants. The effects of dietary PIs in insects are reviewed, and the physiological mechanisms resulting in growth depression in vertebrates are discussed. Chapter two presents a simple, but powerful method to measure quantitatively activities of a wide range of serine proteinase inhibitors using a radial diffusion assay. The assay can detect as little as 2-20 pmol PI, the error is between 4-12%, and the detection range can vary by three orders of magnitude. In chapter three the induction of endogenous PIs in response to insect attack is compared to the response after mechanical wounding and virus infection. It is demonstrated that local induction of PIs after insect attack is very strong in mature tobacco and tomato plants, but that systemic induction is virtually absent. Instead of direct systemic PI induction, it is observed that wounding several leaves at once, creates locally a stronger wound response. This suggests the presence of a systemic factor, which regulates the strength of the local wound response by silent alarm. Chapter four describes the induction of proteinase activity insensitive to plant PIs in the gut of Spodoptera exigua larvae, when the insects are feeding on tobacco leaves containing either potato PI2 or endogenous tobacco PIs It is demonstrated that PIs decrease the proteinase activity in larval guts, but that this reduction is partially compensated for by the induction of PI-insensitive proteinase activity. The weight of larvae, fed with PI leaves, was not reduced, so that the induced PI-insensitive activity, apparently sufficiently, compensated proteinase activity lost by inhibition. Chapter five describes the analysis of gut proteinase activity of S. exigua larvae. Six major proteinase activities were identified and three were purified by anion exchange chromatography and further analyzed. One of the purified proteinases was a cysteine proteinase with optimal activity at pH 11 and is the first example of this class of proteinase to be isolated from a lepidopteran insect. Chapter six demonstrates that potato P12 can be displayed as a functional protein on M13-phages by fusion to a minor coat protein. It is shown that functional PI2-phages mixed with non-functional phages can be enriched 323,000-fold against trypsin after three selection rounds. Large engineered phage libraries of PI2-variants allow the selection of PI2 clones with high affinity for PI-insensitive proteinases; of insect pests. Finally, chapter seven discusses the results comprehensively to defend the thesis that insects acquire resistance against the PIs induced in their host plants. It is argued that the successful application of PIs for resistance breeding will require the selection of better PI genes and that phage display offers a suitable method for this purpose.