dsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine PDF

Preface

The purpose of dsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry and Medicine is to compile and unify current knowledge on the biology of dsRNA moieties from yeast, filamentous fungi, nematodes, plants, and animals. The application of recombinant DNA techniques to dsRNA research has led to significant conceptual advances and paved a path for exciting technical developments and potential solutions to problems ranging from plant disease management to treating genetic conditions in humans. In the last few years, dsRNA research has opened new vistas in biology and offered new tools for studying gene expression and gene function.

dsRNA evokes a multitude of responses in a wide range of organisms ranging from protozoa to vertebrates. The dsRNA-activated protein kinase (PKR), expressed in plants and higher vertebrates, elicits the interferon antiviral and antiproliferative response, may activate the cell death program (apoptosis), and may have supporting roles in cytokine signaling and the immune response, cell differentiation, and transcriptional induction of dsRNA-regulated genes (see Chapter 2).

In contrast, recent studies have shown that dsRNA disrupts gene expression in a sequence-specific manner in several species of invertebrates and in young embryos of lower and higher vertebrates. In these experiments, the introduced exogenous dsRNA targets directly or indirectly the homologous cellular mRNA and activates a mechanism leading to its degradation (see Chapter 1). This phenomenon, called RNA interference (RNAi), is very similar to cosuppression or post-transcriptional gene silencing (PTGS) observed in plants. Interestingly, recent experimental evidence suggests that RNAi and PTGS may employ similar mechanisms. This dsRNArelated research has led to the development of a new, powerful reverse genetics tool that can be used to analyze gene function in a wide range of organisms (see Chapter 1). Furthermore, this technology can be adopted for many commercial applications in agriculture and medicine. In Chapter 1, you will find an outline of potential benefits as well as risks involved in the use of dsRNA-induced gene silencing in different groups of organisms.

As noted above, the dsRNA-induced PKR is involved in the interferon-induced antiviral response and may play important roles in response to cytokines and cellular stress, transcriptional activation, cell growth, regulation of cell differentiation, and cell death (apoptosis) (see Chapter 2). Thus, it is important that these PKR-induced responses be taken into consideration in sequence-specific gene-silencing studies using RNAi technology (see Chapter 1).

The dsRNA viruses from yeast ( Saccharomyces cerevisiae ) and the corn smut incitant, Ustilago maydis , and their killer toxins have been well characterized and can serve as models for the molecular characterization of other dsRNA mycoviruses or dsRNA elements. Moreover, studies on translation of yeast killer dsRNA and

post-translational processing have resulted in the discovery of numerous host (yeast) genes involved in the above as well as other cellular processes (see Chapter 3). In addition, investigations on the mode of action of killer toxins have led to the identification of genes involved in the biogenesis of cell wall. Finally, knowledge gained through studying killer toxins is currently being used to develop new approaches to treating yeast infections or to exclude yeast contaminants in fermentation.

Cloning of the U. maydis preprotoxin genes KP4 and KP6 and their expression in tobacco and maize led to tantalizing findings. The preprotoxins were processed to their respective functional forms identical to those of U. maydis , strong evidence of the existence of the kex2p protease pathway in plants. Furthermore, transgenic maize plants producing KP4 or KP6 toxins were resistant to U. maydis infection (see Chapter 4).

Since the discovery of virus-like particles in diseased mushrooms in 1962, scientists have shown that dsRNAs are ubiquitous in the fungal kingdom. Although fungal dsRNAs often occur in high concentrations, they do not always elicit an overt pathology in their fungal host. Perhaps this is one of the reasons why fungal dsRNA research has remained under-appreciated. Chapters 5 through 10 present cases in which direct or strong indirect experimental evidence has been presented showing that particular dsRNAs are associated with a wide range of biological responses. Chapters 5 through 9 focus on the most recent advances on dsRNA affecting filamentous fungi. The main reason for studying these dsRNAs is to understand how they or their products interfere with normal biological processes in their respective fungal hosts. To date, plant disease management relies heavily on the use of chemicals, leading to increased production costs, utilization of nonrenewable resources, water pollution, non-target effects, and development of tolerance by the target organism. Thus, understanding of genetic factors such as virulence-modulating fungal dsRNAs may lead to the development of biocontrol- or plant-genetic engineeringbased strategies of plant disease management that fulfill the need for sustainable and nonpolluting agricultural practices.

Although most of the dsRNA systems described in Chapters 5 through 9 have been studied well, there remain technical problems related to launching the dsRNA from a cDNA clone. The hypovirus system of Endothia parasitica , the chestnut blight incitant, is the most highly developed dsRNA system of a plant pathogenic fungus. One of the main reasons for this progress has been the availability of a transfection system that launches the dsRNA from an infectious clone. This gene transfer system allowed the unravelling of the mechanism underlying the hypovirusinduced attenuation of virulence in E. parasitica , characterization of virus-encoded determinants responsible for altering the host genotype, and identification of host genes that are up- or down-regulated upon introduction of hypovirus dsRNA (see Chapters 5 and 6). Furthermore, transgenic hypovirulent strains of E. parasitica possess chromosomally integrated hypovirus cDNA and the derived, cytoplasmically replicating dsRNA. Thus, in contrast to naturally occurring hypovirulent strains that produce high percentages of dsRNA-free conidia and 100% dsRNA-free ascospores, essentially all of the spores produced by transfected strains have the integrated cDNA and the corresponding hypovirus dsRNA (see Chapter 5). This property is expected o facilitate dissemination of hypovirulent inoculum and enhance the potential for biological control of the pathogen that devastated both the American and European chestnut in the beginning of the 20th century.

As noted above, lack of an effective gene transfer system has been a major obstacle in using the analytical power of reverse genetics in most of the dsRNA/plant pathogenic fungal systems. Recently, however, a novel gene transfer method involving Agrobacterium tumefasciens has been employed to efficiently transform yeast and several filamentous fungi (see Chapter 10). It is hoped that adoption of agrotransformation will allow development of practical gene transfer methods for several other fungi and, in turn, accelerate unveiling of the biological roles of dsRNAs found in these organisms.

Throughout the pages of this book, you will find examples of dsRNAs associated with perturbation of biological processes in their respective fungal hosts. In one of these cases, a hypovirulence-associated dsRNA (M2) converts a "disposable" pathway, the quinate pathway, from inducible to constitutive (see Chapter 8). The quinate pathway shares two intermediate substrates with the shikimate pathway, which leads to the synthesis of the three aromatic amino acids and other important metabolites. Thus, converting the quinate pathway to constitutive down-regulates the shikimate pathway and converts the host ( Rhizoctonia solani ) to hypovirulent. Quinate is one of the most prevalent phenolic compounds in composted leaf or bark litters and is used as a carbon source by soil bacteria and fungi. Interestingly, quinate converts a virulent, M2-lacking isolate of R. solani to hypovirulent and concurrently induces transcription and translation of a M2-specific gene. More importantly, the quinateinduced hypovirulence and M2 dsRNA expression persist even in the presence of virulence-enhancing amendments such as intermediates of the shikimate pathway or aromatic amino acids. Is it possible that quinate signals switching from parasitism to saprophytism in R. solani and perhaps other soil-borne plant pathogens? If so, would mature, quinate-rich leaf or bark compost become an effective biocontrol medium in the foreseeable future?

Plants are known to harbor bipartite, encapsidated, dsRNA-containing, symptomless cryptic viruses. In addition, several plant species have large-sized, cytoplasmic dsRNAs sequestered within membranous vesicles (see Chapter 11). These dsRNAs cause no visible symptoms and are transmitted only in a vertical manner, and their number of copies per cell is regulated by the nuclear background and developmental stage of the host cell. One of these large dsRNAs causes cytoplasmic male sterility (CMS) in broadbean ( Vicia faba ). Yield quality and quantity of this crop plant are low but could be improved by hybrid breeding, which depends on the availability of male sterile parental lines to prevent self-fertilization. Currently, the dsRNA-associated CMS in broadbean is generally unstable and gives rise to spontaneous revertants. It is tempting to speculate that practical methods will soon be developed for the creation of stable dsRNA-derived CMS in broadbean and other important crops.