Sustainability of insect resistance management strategies for transgenic Bt corn

Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies. An arena ripe for application of such analysis is the deployment of transgenic crops. The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content. However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance. The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act. Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B. thuringiensis (Bt) insecticidal proteins.     

Chemical basis of host-plant resistance to aphids

Abstract The importance of host-plant resistance of crop plants in the biological control of aphids and the formation of aphid biotypes is discussed. The rapid response of plant breeders to the formation of new aphid biotypes requires detailed knowledge on the mechanism of host-plant resistance. Current knowledge on the chemical basis of host-plant resistance to aphids is summarized. Of central importance to this relationship is the aphid-pectinase plant-pectin interdependence. Parallels between the mechanism of aphid-plant and pathogen-plant attack are pointed out and how aphid bacterial endo-symbiotes play a central role in host-plant selection.     

Deterministic in contrast to stochastic modeling.

The first attempt to model a process is often a deterministic setup with differential equations. The existing stochastic influence is suppressed and hopefully negligible. However, sometimes the stochastic component is important. We demonstrate and clarify this for a growth process. The deterministic approach is given by Yn + 1 = Yn + g(Yn) or dYt = g(Yt)dt, Y0 = 1, g a positive function. The corresponding stochastic equation is Xn + 1 = Xn + g(Xn)(1 + xi n) or dXt = g(Xt)dt + f(Xt)dWt, xi some random variable, W the Brownian motion. We compare the asymptotic behavior of the deterministic solution versus the stochastic solution.     

Interaction of fertilizer regime with host-plant resistance in tomato

The effect of fertilizer regime on trichome- and lamellar-based resistance in the wild tomato species, Lycopersicon hirsutum f. glabratum C.H. Mull accession PI 134417, to three insect pests of tomato, the tobacco hornworm, Manduca sexta (L.), the colorado potato beetle, Leptinotarsa decemlineata (Say), and the tomato fruitworm, Heliocoverpa zea (Boddie), was examined. Increasing the rate at which NPK fertilizer was applied, from 1.8 to 19.6 g/plant/week, reduced the trichome-based resistance of PI 134417 to M. sexta and L. decemlineata by lowering both the density of type VI (sensu Luckwill, 1943) glandular trichomes and the amount of 2-tridecanone contained in the tips of these trichomes. 2-Tridecanone is a toxic methyl-ketone responsible for glandular trichome-mediated resistance in PI 134417 to M. sexta and L. decemlineata. A similar increase in the application rate of NPK fertilizer reduced the lamellarbased resistance of PI 134417 to L. decemlineata and H. zea. The meachanisms for this reduction of resistance are unknown, but may be related to improved nutritional quality of hosts at higher fertilizer regimes.     

Tetracycline-inducible Expression Systems： New Strategies and Practices in the Transgenic Mouse Modeling

To accurately analyze the function of transgene(s)of interest in transgenic mice,and togenerate credible transgenic animal models for multifarious human diseases to precisely mimic human dis-ease states,it is critical to tightly regulate gene expression in the animals in a conditional manner.The abilityto turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedentedflexibility in dissecting gene functions in health and disease.Pioneering studies in conditional transgene ex-pression have brought about the development of a wide variety of controlled gene expression systems,whichmeet this criterion.Among them,the tetracycline-controlled expression systems(e.g.Tet-off system andTet-on system)have been used extensively in vitro and in vivo.In recent years,some strategies derived fromtetracycline-inducible system alone,as well as the combined use of Tet-based systems and Cre/lox P switch-ing gene expression system,have been newly developed to allow more flexibility for exploring gene functionsin health and disease,and produce credible transgenic animal models for various human diseases.In thisreview these newly developed strategies are discussed.     

西花蓟马对吡虫啉的抗性机制及交互抗性研究

为了解西花蓟马Frankliniella occidentalis(Pergande)对吡虫啉的抗性风险,本文就吡虫啉的交互抗性和抗性机制(增效剂和酶活性)进行了研究。结果表明,经过35代筛选,西花蓟马对吡虫啉的抗性上升到21.26倍。西花蓟马对吡虫啉与阿维菌素和甲维盐存在中等水平交互抗性,与氯氟氰菊酯、灭多威和毒死蜱存在低水平交互抗性。增效剂试验表明,三丁基三硫磷酸酯(DEF)、磷酸三苯酯(TPP)和马来酸二乙酯(DEM)具有显著增效(SR50,DEF=6.38,SR50,TPP=5.52,SR50,DEM=1.60,P<0.05)。生化测定表明:抗吡虫啉品系西花蓟马的羧酸酯酶(5.06倍)和谷胱甘肽S-转移酶酶活性(1.63倍)均显著(P<0.05)高于敏感品系,表明羧酸酯酶和谷胱甘肽S-转移酶酶活性的提高是西花蓟马对吡虫啉产生抗药性的主要原因。     

Risk assessment strategies for transgenic plants

Advances in recombinant DNA technology have created advantages for the development of plants with high agro-economical values. Since the production of transgenic plants, some issues concerning the safe use of these plants and their products have been under debate throughout the world. In this respect, the potential risks and benefits of transgenic plants need to be evaluated objectively. Risk assessment of transgenic crops is a basic prerequisite for monitoring the possible risks that could arise upon the release and use of transgenic plants. To get a meaningful tool for decision making, risk assessment needs to be carried out in a scientific sound and transparent manner. There are specific governmental regulations in many countries for the safety assessment of genetically modified (GM) crops. Furthermore, there are some international agreements, which regulate the cultivation and commercialization of transgenic plants and their derivatives. Internationally accepted risk assessment strategies have been performed to evaluate the safe use of a large variety of GM crops. The main objectives of these regulations and risk assessment strategies are focused to protect human/animal health and the environment.     

Expression of a dominant negative SHP-2 in transgenic mice induces insulin resistance.

To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (DeltaPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that DeltaPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues.     

Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.     

The ecology and evolution of host-plant resistance to insects

Genetic techniques have yielded new insights into plant-herbivore coevolution. Quantitative genetic tests of herbivory theory reveal that in some cases insect herbivores impose selection on resistance traits. Also, some resistance traits are costly while others appear not to be, and genetic models can explain these results. Genetic variation in plant resistance influences insect community structure by modifying interactions of herbivores with competitors and natural enemies. Therefore, models of multispecies coevolution are more realistic than pairwise coevolutionary models. Ecological genetics will facilitate further theoretical and empirical exploration of multispecies coevolution of plants and herbivores.     

Molecular strategies for gene containment in transgenic crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.     

Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

Evaluating genetic containment strategies for transgenic plants

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape into the environment. The likelihood that a transgene will spread in the environment depends on its potential fitness impact. The fitness conferred by various transgenes to crop and/or wild-type hybrids has been evaluated in several species. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops.     

Cadmium-induced adaptive resistance and cross-resistance to zinc in Xanthomonas campestris

Cadmium (Cd) and zinc (Zn) are environmental pollutants affecting both soil and water. The toxicity resulting from the exposure of Xanthomonas campestris, a soil bacterium and plant pathogen, to these metals was investigated. Pretreatment of X. campestris with sub-lethal concentrations of Cd induced adaptive protection against subsequent exposure to lethal doses of Cd. Moreover, Cd-induced cells also showed cross-resistance to lethal concentrations of Zn. These induced protections required newly synthesized proteins. Unexpectedly, Zn-induced cells did not exhibit adaptive protection against lethal concentrations of Zn or Cd. These data suggested that the increased resistance to Cd and Zn killing probably involved other protective mechanisms in addition to ion efflux.     

Phenotyping transgenic wheat for drought resistance

Realistic experimental protocols to screen for drought adaptation in controlled conditions are crucial if high throughput phenotyping is to be used for the identification of high performance lines, and is especially important in the evaluation of transgenes where stringent biosecurity measures restrict the frequency of open field trials. Transgenic DREB1A-wheat events were selected under greenhouse conditions by evaluating survival and recovery under severe drought (SURV) as well as for water use efficiency (WUE). Greenhouse experiments confirmed the advantages of transgenic events in recovery after severe water stress. Under field conditions, the group of transgenic lines did not generally outperform the controls in terms of grain yield under water deficit. However, the events selected for WUE were identified as lines that combine an acceptable yield-even higher yield (WUE-11) under well irrigated conditions-and stable performance across the different environments generated by the experimental treatments.     

Potential use of theaux2 gene fromAgrobacterium rhizogenes as a conditional negative marker in transgenic cabbage

An originalAgrobacterium tumefaciens-mediated transformation procedure, based on the actions of both wild type and disarmed bacterial strains, was developed. Theaux2 gene ofA. rhizogenes was introduced into a rapid-cycling genotype of cabbage (Brassica oleracea L.). Theaux2 gene product converts naphthalene acetamide into the auxin naphthalene acetic acid. Expression of this gene in the transgenic progeny grownin vitro led to an altered root phenotype. On a medium supplemented with napthalene acetamide (NAM), two of the three analysed progenies were characterized by the formation of callus instead of roots, whereas on a NAM-free medium all the plantlets from these progenies presented a normal phenotype. Expression of theaux2 gene was also assessed under horticultural conditions by sowing seeds in sand and watering them with a nutritive solution supplemented with NAM. Under these conditions, NAM inhibited the formation of a root system in transgenic plantlets and induced the death of the transgenic plantlets three to four weeks after germination. Thus,aux2 acts as a lethal conditional marker which could be used in negative selection of cabbage. Potential utilization of theaux2 gene to screen spontaneous androgenetic plants in order to transfer cytoplasmic male sterility in a single generation is discussed.     

Substantiation in Enterococcus faecalis of dose-dependent resistance and cross-resistance to pore-forming antimicrobial peptides by use of a polydiacetylene-based colorimetric assay

A better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and for in vitro assessment, variants of Enterococcus faecalis that are resistant to different doses of the fungal AMP alamethicin (Alm(r)) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Alm(r) variants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Alm(r) variants showed cross-resistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Alm(r) variants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using an in vitro colorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.     

Effect of bioregulator-treated sorghum on greenbug fecundity and feeding behavior: implications for host-plant resistance

Three commercial and six experimental plant growth bioregulators were surveyed for their effect on aphid reproduction when applied to sorghum. Only CCC and PIX had a significant effect on the greenbug, Schizaphis graminum. Application of the commercial bioregulators CCC and PIX^R caused about a 50% decrease in aphid reproduction rate when applied to greenbug susceptible sorghum but had little effect when applied to a greenbug resistant sorghum line. Electronic monitoring of aphid probing behavior on CCC treated, greenbug-susceptible sorghum showed a response pattern which was indistinguishable from that normally observed on greenbug resistant lines and was different from that associated with aphid probing behavior on untreated susceptible lines. The isolated pectin content of the CCC treated susceptible sorghum was twice that of the controls and had twice the methoxy content. These results support the argument that pectin is a barrier to aphid-stylet penetration for phloem feeding aphids which probe intercellularly and that manipulation of pectin content and/or structure can be a major factor in host-plant resistance to sap-sucking insects.     

The use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies

This review compares the activity of the plant transposable elements Ac, Tam3, En/Spm and Mu in heterologous plant species and in their original host. Mutational analysis of the autonomous transposable elements and two-element systems have supplied data that revealed some fundamental properties of the transposition mechanism. Functional parts of Ac and En/Spm were detected by in vitro binding studies of purified transposase protein and have been tested for their importance in the function of these transposable elements in heterologous plant species. Experiments that have been carried out to regulate the activity of the Ac transposable element are in progress and preliminary results have been compiled. Perspectives for manipulated transposable elements in transposon tagging strategies within heterologous plant species are discussed.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.