Transgenic Plants with Enhanced Resistance to the Fungal Pathogen Rhizoctonia solani.

The production of enzymes capable of degrading the cell walls of invading phytopathogenic fungi is an important component of the defense response of plants. The timing of this natural host defense mechanism was modified to produce fungal-resistant plants. Transgenic tobacco seedlings constitutively expressing a bean chitinase gene under control of the cauliflower mosaic virus 35S promoter showed an increased ability to survive in soil infested with the fungal pathogen Rhizoctonia solani and delayed development of disease symptoms.

Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

Activation of a Bean Chitinase Promoter in Transgenic Tobacco Plants by Phytopathogenic Fungi.

The temporal and spatial expression of a bean chitinase promoter has been investigated in response to fungal attack. Analysis of transgenic tobacco plants containing a chimeric gene composed of a 1.7-kilobase fragment carrying the chitinase 5B gene promoter fused to the coding region of the gus A gene indicated that the chitinase promoter is activated during attack by the fungal pathogens Botrytis cinerea, Rhizoctonia solani, and Sclerotium rolfsii. Although induction of [beta]-glucuronidase activity was observed in tissues that had not been exposed to these phytopathogens, the greatest induction occurred in and around the site of fungal infection. The increase in [beta]-glucuronidase activity closely paralleled the increase in endogenous tobacco chitinase activity produced in response to fungal infection. Thus, the chitinase 5B-gus A fusion gene may be used to analyze the cellular and molecular details of the activation of the host defense system during pathogen attack.

Frequency-Dependent Stability for Two-Species Interactions

Evolutionary game theory is extended to models of two-species interactions where fitnesses are based on individual characteristics (strategies) rather than on a population dynamic that assumes homogeneous species. It is shown that the coevolutionary theories in the literature that combine ecology with genetic viability selection are part of this extended theory and that the dynamic stability resulting from a separation of ecological and evolutionary processes actually follows from game-theoretic solution concepts. The main focus of the paper is to investigate the application of the ESS (evolutionarily stable strategy) solution concept to dynamic stability when fitnesses are given by random interactions between individuals as opposed to viability selection. For two-species frequency-dependent interactions, the ESS criterion that implies stability asserts that, in any system near the ESS, at least one of the species is better off (i.e., more fit) if it evolves towards the ESS. The global stability of a polymorphic two-species ESS that is shown for two-species matrix games gives a powerful tool to predict the course of evolution through static fitness comparisons.

A 7 year experience with low anterior sigmoid resections using the EEA stapler.

The EEA stapler was used in 205 patients over a 7 year period with 19 complications (9 percent) and 1 death (0.5 percent). Meticulous attention to technique and familiarization with the EEA stapler are necessary to achieve such results. The use of mechanical and oral antibiotic bowel preparation, perioperative systemic antibiotics, povidone-iodine irrigation of the rectal ampulla, and meticulous surgical technique have combined to lower the complication rate of this procedure to a minimum. The EEA stapler has greatly improved the safety and ease of anterior sigmoid resections and now provides a frequent alternative to abdominoperineal resection in many patients with midrectal and even low rectal lesions.

Evolutionarily stable sets in symmetric extensive two-person games.

Evolutionarily stable (ES) sets are characterized for evolutionary games in extensive form. It is shown that, for the normal form of games involving informational symmetries or repeated play, the standard approach of determining evolutionarily stable strategies (ESSs) often fails to predict the evolutionary outcome. The dynamic stability of ES sets is proved in both the pure strategy and mixed strategy models. ES sets are shown to also generalize the notion of direct ESSs (an earlier attempt to apply ESS theory to extensive games). The theory is illustrated by three examples of biological games in extensive form.

Strong stability and density-dependent evolutionarily stable strategies.

Stability conditions for equilibria of the evolution of population strategies in a single species are developed by comparing frequency and density dependent fitnesses of pairs of strategies. Stability of such equilibria is shown for general haploid frequency and density dynamics. It is also shown that this stability is stronger than that of multispecies population dynamical systems. A biological interpretation of the conditions is provided in terms of the fitness of invading subpopulations.

Frequency-dependent viability selection (a single-locus, multi-phenotype model).

Frequency-dependent natural selection models are examined where the viability of an individual in the diploid population is determined by its phenotype and the frequency of other phenotypes present. The equilibria of the multi-phenotypic system are characterized through local mean fitness functions. It is shown that stability can best be analyzed by combining the principles of maximization of population mean fitness with the evolutionary stability conditions that apply when phenotypic fitnesses relative to the genetic constraints are equal.

Complex dynamical behaviour of frequency-dependent viability selection: an example.

The model of viability selection based on a one-locus, two-allele diploid population is considered. Frequency dependence is introduced through the fitnesses of three phenotypes (strategies) exhibited by the population. Both discrete and continuous dynamics are analyzed and contrasted with the classical results of frequency-independent selection and with the more recent results of frequency-dependent selection based on two-phenotype multi-allele systems. Cycling and chaotic behaviour are shown to be easily obtained in the discrete model. Intuitive biological conditions for stability are shown to fail as well as at general equilibria of the continuous model.

Evolutionary stability concepts for N-species frequency-dependent interactions.

The classical static concept of an evolutionarily stable strategy (ESS) for a single species gives rise to two new notions when there are more than two species (called an N-species ESS and RL-stability). The paper relates these to the dynamic stability of monomorphic and polymorphic evolutionary systems. It is shown that RL-stability implies the global asymptotic stability of either system with or without mutations. However, the N-species ESS only implies stability of the monomorphic system.

Spatial and density effects in evolutionary game theory.

Two models are considered for the study of game dynamics in a spatial domain. Both models are continuous in space and time and give rise to reaction-diffusion equations. The spatial domain is homogeneous but the mobility of the individuals is allowed to depend upon the strategy. The models are analysed for spatial patterns (via a Turing instability) and also for the direction of the travelling wave that replaces one strategy by another. It is shown that the qualitative behaviour of the two models is quite different. When considering the existence of spatial patterns and deciding whether increased mobility is helpful or not, it is shown that the answers depend crucially upon the model equations. Since both models (in the absence of spatial variation) are quite standard, it is clear that considerable care has to be exercised in the formulation of spatial models and in their interpretation.

Nonlinear frequency-dependent selection at a single locus with two alleles and two phenotypes.

The paper investigates the discrete frequency dynamics of two phenotype diploid models where genotypic fitness is an exponential function of the expected payoff in the matrix game. Phenotypic and genotypic equilibria are defined and their stability compared to frequency-dependent selection models based on linear fitness when there are two possible phenotypes in the population. In particular, it is shown that stable equilibria of both types can exist in the same nonlinear model. It is also shown that period-doubling bifurcations emerge when there is sufficient selection in favor of interactions between different phenotypes.

An ESS maximum principle for matrix games.

Previous work has demonstrated that for games defined by differential or difference equations with a continuum of strategies, there exists a G-function, related to individual fitness, that must take on a maximum with respect to a virtual variable v whenever v is one of the vectors in the coalition of vectors which make up the evolutionarily stable strategy (ESS). This result, called the ESS maximum principle, is quite useful in determining candidates for an ESS. This principle is reformulated here, so that it may be conveniently applied to matrix games. In particular, we define a matrix game to be one in which fitness is expressed in terms of strategy frequencies and a matrix of expected payoffs. It is shown that the G-function in the matrix game setting must again take on a maximum value at all the strategies which make up the ESS coalition vector. The reformulated maximum principle is applicable to both bilinear and nonlinear matrix games. One advantage in employing this principle to solve the traditional bilinear matrix game is that the same G-function is used to find both pure and mixed strategy solutions by simply specifying an appropriate strategy space. Furthermore we show how the theory may be used to solve matrix games which are not in the usual bilinear form. We examine in detail two nonlinear matrix games: the game between relatives and the sex ratio game. In both of these games an ESS solution is determined. These examples not only illustrate the usefulness of this approach to finding solutions to an expanded class of matrix games, but aids in understanding the nature of the ESS as well.

Evolutionary games and two species population dynamics.

Competition between species has long been modeled by population dynamics based on total numbers of each species. Recently, the evolution of strategy frequencies has been used successfully for competition models between individuals. In this paper, we illustrate that these two views of competition are compatible. It is shown that the rate of intra and interspecific competitions between individuals largely determines the population dynamics. Competition models over a single common resource and predator-prey models are developed from this individual competition approach. In particular, the equilibrium strategies in a co-evolving predator-prey system are shown to be more stable than the predicted strategy cycling of standard evolutionary game theory.

Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco.

Expression of at least two genes from bean encoding the defense-related protein chitinase has been shown previously to be transcriptionally regulated by the phytohormone ethylene. We have determined the complete nucleotide sequence of one of these genes, the CH5B gene, which resides on a 4.7-kilobase fragment of bean genomic DNA. The structural gene consists of a single open reading frame and encodes the 301 amino acids of the mature protein and a 26-amino acid signal peptide. The CH5B gene has been introduced into tobacco plants using Agrobacterium Ti-plasmid vectors. Little or no expression of the bean gene was observed when transgenic tobacco plants were grown in air; however, exposure of these plants to an atmosphere containing 50 parts per million ethylene resulted in an approximately 20-fold to 50-fold increase in the level of the bean chitinase mRNA. Ethylene-dependent expression of a chimeric gene consisting of 1.6 kilobases of 5'-flanking DNA derived from the CH5B gene fused to the coding sequence of beta-glucuronidase indicates that this region of the CH5B gene is sufficient for ethylene-regulated expression. Deletion analysis of the CH5B promoter region has allowed us to localize these DNA sequences to within a 228-base pair region situated between -422 and -195 upstream of the transcriptional start site. This region is characterized by two short DNA sequences that are exactly conserved in a second ethylene-regulated bean chitinase gene.