Genetic engineering strategies for enhancing tomato resistance to fungal and bacterial pathogens

The classification and detailed overview of the currently known effective strategies used to increase the resistance of tomato (Solanum lycopersicum L., syn. Lycopersicon esculentum Mill.) plants to infectious fungal and bacterial diseases by genetic engineering approaches are presented. Modern data on the mechanisms of the protective effect of heterologous genes on the enhancement of transgenic tomato resistance to fungal and bacterial pathogens are discussed.     

Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.     

Conserved fungal genes as potential targets for broad-spectrum antifungal drug discovery

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.     

Application of recombinant DNA technology to plant protection: molecular approaches to engineering virus resistance in crop plants

Developments in plant tissue culture, plant transformation and regeneration, and improvements in techniques to isolate and manipulate viral genes have led to the exploitation of the concept of cross protection: turning the virus onto itself and controlling it with its own genes. By introducing and expressing genes of viral origin in crop plants, scientists have engineered resistance to several plant viruses. Some of the approaches, used singly or in combination, include expression of viral-coat protein, untranslatable sense or antisense RNA, satellite RNA, virusspecific neutralizing antibody genes, plant viral replicase, protease or movement proteins and defective, interfering RNA. All of these approaches have resulted in manifestation of virus resistance to varying degrees in several commercially important crop plants. This review summarizes the recent advances in engineering virus resistance using the above approaches, and lists specific examples of their use in cultivated crop plants of economic importance.H.R. Pappu and C.L. Niblett are with the Plant Pathology Department, University of Florida. Gainesville, FL 32611-0680, USA; R.F. Lee is with the Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA. Florida Agricultural Experiment Station Journal Series No. R-04558.     

Genetic analysis of osmotic adjustment in crop plants

Plant water deficit is a component of several different stresses, including drought, salinity and low temperatures, which severely limit plant growth and crop productivity. Genetic modification of plants to allow growth and yield under unfavourable conditions is an important component of the solution to problems of environmental stress. While disagreement and even confusion may characterize some of the discussions on what constitutes a significant and an effective osmotic adjustment (OA) is receiving increasing recognition as a major mechanism. This paper starts with review of OA functions, genetic variation and inheritance, and theories and principles involved in commonly used protocols for quantifying OA. Emphasis is placed on a summary of current molecular strategies and advanced in the improvement of plant stress resistance through manipulating OA. They include a genetic engineering approach and a QTL mapping approach. Future promising strategies for improving drought resistance lie in molecular technology that allows genes or QTLs controlling OA to be tagged and isolated, these genes to be expressed in transgenic plants, and efficiency of breeding via marker-assisted selection to be improved. Aspects of QTL utilization in plant genetics, breeding and physiology and future research directions are discussed.     

Genetic engineering for removing food allergens from plants

Genetic engineering has great potential for improving the safety of plant-based foods by eliminating allergenic components. A recent publication by Dodo et al. demonstrates that RNA interference can mediate the silencing of a major allergen protein from peanuts. Three additional papers (two by Le et al. and one by Lorenz et al.) report the removal of allergenic proteins from tomato fruits. This research highlights the potential for using genetically engineered hypoallergenic plants in a new approach to alleviating food allergy symptoms.     

Genetic engineering of forest woody plants

The present state of genetic engineering (GE) of forest woody plants is considered with special reference to the materials of the International Conference "Wood, Breeding, Biotechnology and Industrial Expectations" held in France in June, 2001. Main tree species subjected to GE are listed, aims of constructing transgenic plants discussed, and methods described. Major achievements in the field are considered along with the problems associated with the employment of GE in the breeding of forest woody plants.     

Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants

As a proof of concept, the qualitative and quantitative engineering of carotenoid formation has been achieved in crop plants. Successful reports in tomato, potato, rice, and canola all describe the enhancement of carotenoid with nutritional value, while in model systems such as tobacco and Arabidopsis the engineering of carotenoid to confer abiotic stress has been described. For all the successful applications there have been many examples of unintended/unpredicted phenotypes and results. Typically this has resided from our lack of understanding of carotenoid formation and its regulation. In the present article, we will review advances in carotenoid formation and its regulation to illustrate how metabolic engineering experiments have shed light on regulatory mechanisms.     

Metabolic engineering of plants for osmotic stress resistance.

Genes encoding critical steps in the synthesis of osmoprotectant compounds are now being expressed in transgenic plants. These plants generally accumulate low levels of osmoprotectants and have increased stress tolerance. The next priority is therefore to engineer greater osmoprotectant synthesis without detriment to the rest of metabolism. This will require manipulation of multiple genes, guided by thorough analysis of metabolite fluxes and pool sizes.     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerpringting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

基因工程与植物的遗传改良

概述了植物基因工程的发展历史及其在植物遗传改良中与常规改良技术相比具有的明显优势,介绍了经基因工程技术改良的转基因植物研究与应用状况,分析了植物基因工程在植物遗传改良中的潜在风险．阐述了利用植物基因工程进行遗传改良与常规遗传改良的关系,并对今后基因工程在植物遗传改良中的应用前景进行了展望。     

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.