Approaches for improving crop resistance to soilborne fungal diseases through biotechnology usingSclerotinia sclerotiorumas a case study

Fungal diseases damage crop plants and affect agricultural production. Transgenic plants have been produced by inserting antifungal genes to confer resistance against fungal pathogens. Genes of fungal cell wall-degrading enzymes, such as chitinase and glucanase, are frequently used to produce fungal-resistant transgenic crop plants. In this review, we summarize the details of various transformation studies to develop fungal resistance in crop plants.

Mechanism Analysis of Broad-spectrum Disease Resistance Induced by Expression of Anti-apoptotic p35 Gene in Tobacco

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61% mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant-pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host-cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant-pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Expression of Bacillus thuringiensis insecticidal protein genes in transgenic crop plants

Switching on Plant Immune Signaling Systems Using Microbe-Associated Molecular Patterns

During the search for potential target genes of WRKY DNA-binding transcription factors, we have previously identified four pathogen-induced Arabidopsis genes (CRK5, CRK6, CRK10 and CRK11) encoding receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. In the present study, we transformed Arabidopsis plants with the RLK genes under control of the constitutive CaMV 35S promoter or a steroid-inducible Ga14 promoter. Expression of CRK5, but not the three other RLK genes, resulted in significant alterations in defense responses and leaf growth in transgenic plants. In transgenic plants harboring the 35S::CRK5 construct, significantly elevated and constitutive expression of CRK5 correlated with enhanced leaf growth and increased resistance to the bacterial pathogen Pseudomonas syringae. The enhanced disease resistance in the transgenic plants was associated with more rapidly induced expression of the PR1 gene after pathogen infection. In transgenic plants transformed with CRK5 under control of the steroid-inducible promoter, expression of the transgene was induced at relatively high levels after the steroid application and this induced expression of CRK5 triggered hypersensitive response-like cell death. Induced CRK5 expression also activated cell death in the npr1, ndr1 and eds1 mutants and in the transgenic nahG plants that fail to accumulate salicylic acid. Thus, the novel RLK is capable of activating multiple distinct defense responses depending on the manner and/or the levels of its over-expression in transgenic plants.

Genetic Engineering of Crop Plants

Transgenic crops expressing insecticidal toxins could soon provide safe, clean and effective means of pest control, but their usefulness will be short-lived if insects adapt to the toxins. Two planting strategies are among those that have been recommended to delay crop failure: susceptible insects could be conserved by planting either ‘refugia’, i.e. separate fields of toxic and toxin-free crop, or ‘seed mixtures’ of toxic and toxin-free plants in the same fields. However, we show that if insects can move from plant to plant, seed mixtures may actually hasten insect resistance compared with pure stands of toxic plants. Insect movement causes an increase in effective genetic dominance which can counteract reduced selection due to the mixture. This failure of seed mixtures is likely under just those conditions, low genetic dominance of resistance, which predict a good chance for resistance to the toxin to evolve slowly. Seed mixtures, unlike refugia, are therefore failure prone. This result also suggests potential problems with a third strategy, tissue-specific expression of toxins, which essentially provides a mixture of toxin-free and toxin-containing tissues on the same plant. However, better information and modelling are urgently required to evaluate alternative means of slowing insect adaptation to resistant crop plants. Legislation for toxinfree refugia may provide one of the best available means for conserving insect susceptibility.

Can plant defensins be used to engineer durable commercially useful fungal resistance in crop plants?

Plant Biotechnology - An Emerging Field. Plant-Derived Drugs and Extracts. Industrial Strategies for the Discovery of Bioactive Compounds from Plants. Plant Cell and Tissue Culture Techniques Used in Plant Breeding. Plant Cell Cultures as Producers of Secondary Compounds. Genetic Transformation of Plants and Their Cells. Properties and Applications of Hairy Root Cultures. Bioreactors for Plant Cell and Tissue Cultures. The Potential Contribution of Plant Biotechnology to Improving Food Quality. Engineering Plant Biochemical Pathways for Improved Nutritional Quality. Transgenic Plants as Producers of Modified Starch and Other Carobhydrates. Improving the Nutritional Quality and Functional Properties of Seed Proteins by Genetic Engineering. Transgenic Plants as Sources of Modified Oils. Flavors and Fragrances from Plants. Fine Chemicals from Plants. Genetic Engineering of the Plant Cell Factory for Secondary Metabolite Production: Indole Alkaloid Production in Catharanthus roseusas a Model. Transgenic Plants for Production of Immunotherapeutic Agents. Signal Transduction Elements. The Plant Cell Wall - Structural Aspects and Biotechnological Developments. Liginin Genetic Engineering: A Way to Better Understand Lignification beyond Applied Objectives. Transgenic Plants Expressing Tolerance Toward Oxidative Stress. Transgenic Plants with Increased Tolerance against Viral Pathogens. Transgenic Plants with Enhanced Tolerance against Microbial Pathogens. Transgenic Crop Plants with Increased Tolerance to Insect Pests. Transgenic Herbicide Resistant Crops - Advantages, Drawbacks and Failsafes. Plants and Environmental Stress Adaptation Strategies. Molecular Mechanisms that Control Plant Tolerance to Heavy Metals and Possible Roles in Manipulating Metal Accumulation. Index.

Engineering salt tolerance in crop plants

Manipulation of Reactive Oxygen Species, Redox and Nitric Oxide Signaling Systems to Activate Plant Innate Immunity for Crop Disease Management

Chapter 1 - Transgenics in Phytoremediation of Metals and Metalloids: From Laboratory to Field

Hydrogen peroxide (H2O2) has been implicated in the induction of plant defense genes and programmed cell death. Expression of a chimeric fungal glucose oxidase (GO) gene driven by a pathogen- and wound-inducible promoter was evaluated in transgenic tobacco and canola as a possible tool for engineering plant cell death and defense gene induction. Expression of this gene under the control of a peroxidase gene promoter resulted in the accumulation of relatively low levels of H2O2 in the young leaves of transgenic tobacco plants and this was not sufficient to cause any visible cell death and defense gene induction as measured by PR-1a mRNA induction. Older leaves of transgenic tobacco plants, however, exhibited visible necrotic lesions and constitutively expressed PR-1a mRNA when grown under high light conditions. Inoculation of cotyledons of control and transgenic canola with Leptosphaeria maculans resulted in rapid cotyledon senescence in the transgenic plants. Strong activators of the peroxidase promoter, i.e., wounding and inoculation of transgenic plants with Cercospora nicotianae, were not sufficient to trigger any additional visible cell death in transgenic tobacco plants, compared with controls. However, when exogenous glucose was supplied to transgenic tissue, massive cell death and PR-1a gene induction were observed in tobacco. Exogenously applied salicylic acid further increased the rate and extent of cell death. Our results suggest that efficacy of GO expression for the induction of cell death is restricted by glucose supply in the plants and are consistent with a role for salicylic acid in the potentiation of plant cell death by H2O2.