Towards protease inhibitor mediated resistance to western flower thrips in chrysanthemum

Dendranthema grandiflora (chrysanthemum) is a cutflower grown across the world, with an acreage similar to roses. Frankliniella occidentalis (Western flower thrips, WFT) is a major pest against a large number of crops, including chrysanthemum, in both field and glasshouse cultivation. Aiming at WFT resistance in chrysanthemum by a transgene-mediated approach, the prerequisite of an efficient regeneration system was first established. Subsequently, promoters for high level transgene expression were identified. The predominant group of proteases active in WFT was identified as the class of cysteine proteases, and the recombinant cysteine protease inhibitors potato cystatin (PC) and equistatin (EI) were shown in vitro to achieve over 90% inhibition of WFT protease activity. PC and EI ingested by adult WFT on a pollen diet for a period of five days resulted in 50% reduction in the oviposition rate. Effects on adult mortality were not observed during this brief period. An eight domain cysteine protease inhibitor potato multicystatin (PMC) was expressed in chrysanthemum in an attempt to endow it with WFT resistance. Low expression levels ranging at maximum 0.1-0.13% of total protein were generated. Hence, no correlation with thrips resistance could be established with these lines. However, the results in this thesis provide evidence of potentially significant effects of cysteine protease inhibitors on WFT oviposition rate. This would have a strong effect on restricting WFT population build up. The concept of enhancing cysteine protease inhibitor activity by various approaches in the different parts of chrysanthemum to combat WFT is discussed in relation to the existing alternatives.</p

Pyrethrins Protect Pyrethrum Leaves Against Attack by Western Flower Thrips, Frankliniella occidentalis

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

Effects of cysteine protease inhibitors on oviposition rate of the western flower thrips, Frankliniella occidentalis

Proteolytic activity in whole insect extracts of the western flower thrips, Frankliniella occidentalis, was found to belong predominantly to the class of cysteine proteases. The pH optimum of the general proteolytic activity was determined to be 3.5, which is low when compared to other insects using cysteine proteases for protein digestion. The proteinaceous cysteine protease inhibitors chicken cystatin, potato cystatin and sea anemone equistatin inhibited in vitro more than 90 f the protease activity. To test in vivo the biological effect of such inhibitors on the oviposition rate of western flower thrips, recombinant potato cystatin and equistatin were fed to adult females. A gradual reduction in oviposition rate to about 45 f control was observed when reared on these PIs for a period of 5 days, with no increase in mortality. These results are discussed in the light of the application of protease inhibitors in transgenic plants to control this insect pest

The potato Lhca3.St.1 promoter confers high and stable transgene expression in chrysanthemum, in contrast to CaMV-based promoters

reporter gene with and without flanking matrix-associated regions (MARs).They were transferred into chrysanthemum viaAgrobacterium-mediated transformation. The quantitativeevaluation of GUS activity in a total of 127 independently derivedtransformantsestablished that in chrysanthemum the Lhca3.St.1 promoterwas 175 fold more active in the leaves than the dCaMV promoter was. The latterwas as poor in expression as the single CaMV promoter. The use of suchCaMV-based promoters in the genetic engineering of chrysanthemum should bediscouraged when high levels of transgene expression are desired. No clearinfluence of the presence of MARs was observed on the variability of GUS geneexpression, in contrast to earlier studies in tobacco. This may indicate apossible plant species dependent activity of MAR elements.Lhca3.St.1 promoter-driven GUS activity was relativelyhigher in the stem of chrysanthemum and proved stable over extensive timeperiods. Therefore this potato promoter is attractive to obtain high expressionlevels in chrysanthemum

Expression of potato multicystatin in florets of chrysanthemum and assessment of resistance to western flower thrips, Frankliniella occidentalis

The gene of the 85 kDa cysteine protease inhibitor (CPI) potato multicystatin (PMC), was PCR-cloned and expressed under the control of the UEP1 promoter in chrysanthemum (Dendranthema grandiflora). Over 30 independent transgenic plant lines were analysed for PMC expression in florets by immunoassay and a selection of those for papain inhibitor activity (PIA). A significant correlation between PIA activity and PMC expression levels was established demonstrating that the highest expressers raised the concentration of PIA to 0.28 pmol/μg from an endogenous background level of 0.15 pmol/μg. On this basis, the PMC gene was estimated to be expressed at a level of 0.13␘f total protein. Some of the transgenic lines exhibited up to 5-fold higher levels of PIA (0.82 pmol/μg protein). This did not correlate with the immunological data, however, and may be the result of frequently occurring somaclonal variation. A non-choice bioassay of 7-10 days on whole flowers was carried out to study the effect of PMC on the fecundity of Western Flower Thrips in terms of the number of larvae produced. No correlation between the reduction in the oviposition rate and PMC expression could be established, which may be due to the relatively low expression level of PMC in chrysanthemum. The transgenic lines with the highest levels of endogenous PIA also had the lowest thrips reproduction rates, but further experiments are required to exclude artefacts caused by the aberrant phenotype

Cloning, Functional Expression in Pichia pastoris, and Purification of Potato Cystatin and Multicystatin

In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1 l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (Ki) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain

Cloning of the chrysanthemum UEP1 promoter and comparative expression in leaves and ray and disc florets of Dendranthema grandiflora

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