The lab exercises enable students to learn about properties of Tobacco mosaic virus (TMV) including (1) symptoms induced by the virus in susceptible plants at the macroscopic and microscopic levels, (2) its stability at high temperatures, and (3) its small size.
Viruses cause a variety of diseases in plants and animals. We are most familiar with those that affect humans, such as measles, mumps, and polio. At one time, these diseases posed major health concerns, but with the widespread use of vaccinations in children, they now occur incidentally. Others, such as the AIDS-causing virus, human immunodeficiency virus (HIV), are currently global concerns.
The study of plant viruses played an important role in the study of viruses since the fundamental nature of viruses was first discovered in a disease of tobacco, now known to be caused by Tobacco mosaic virus (TMV). Since this initial discovery a little over a century ago, more than 900 plant viruses have been described.
In several regions in Europe during the nineteenth century, a disease significantly curtailed tobacco production because of a distortion of the leaves and a reduction in yield and quality. The disease, known as tobacco mosaic, was named for the abnormal mixture of green, pale green, and yellow colors (or mosaic) on the leaves (Figure 1). Since little was known about what caused the disease, scientists tried to identify the pathogen using the common methods for isolating bacterial and fungal pathogens, but their attempts were unsuccessful.
|Figure 1. Symptoms of Tobacco mosaic virus in tobacco. Note the narrowed and mottled or mosaic appearance of the leaves. (Courtesy H.D. Shew). Click image to see a larger view.
During the time these early studies were conducted, scientists believed that all pathogens were organisms composed of one or more cells (e.g., bacteria, fungi, and nematodes). This belief was an extension from the cell theory, proposed by Schleiden and Schwann in 1839, that all living things, which would include pathogens, were made of cells. A surprising discovery from these early investigations was that the pathogen of the tobacco disease proved to be smaller than a cell. The evidence leading to this realization came from the observation that sap from infected plants that was passed through a bacteria-proof filter was still able to infect plants. M. W. Beijerinck, a professor of microbiology from the Netherlands, called it a contagium vivum fluidum or contagious living fluid. Later, the liquid that passed through the filter came to be known simply as a virus, which in Latin means poison or slime. For many years, viruses were recognized as the smallest entity known to cause disease. But today, pathogens are known that are composed of only a single small RNA molecule (a viroid) or a protein (a prion).
Other scientific advancements can also be attributed to experimentation with TMV. It was the first virus to be chemically purified. Its particle structure was one of the first described, and its genome or gene-containing nucleic acid was one of the first to be sequenced. The study of TMV, though, is not merely of historical significance; today many scientists are learning more about molecular interactions between a virus and its host using this well studied virus.
The virus: TMV is the type or common member of the genus Tobamovirus. The particles or virions are rod-shaped with an average length of 300 nm and width of 18 nm (Figure 2A). The virus is a complex macromolecule made of RNA (5%) and protein (95%). Its single molecule of RNA is about 6400 nucleotides in length and contains the genetic information of the virus. The protein surrounds the entire length of the RNA and protects the RNA from degradation both inside and outside of the cell. These coat proteins, as they are called, bind to the entire length of the RNA (Figure 2B).
|Figure 2. An electron micrograph of Tobacco mosaic virus particles (A), and a model of the virus illustrating the arrangement of the RNA and its coat protein (B). (Courtesy APS). Click image to see a larger view.
The viral genes: The RNA codes for four genes (Figure 3). Two of these are necessary for the replication of the viral nucleic acid (called replicase-associated proteins); one is for the cell-to-cell movement within the plant (movement protein); the last one codes for the coat protein, the only protein that becomes a part of the virus particle.
|Figure 3. The genetic map of Tobacco mosaic virus. The positions of the four genes are shown. The two larger ones (replicase-associated proteins) (A and B) code for proteins that participate in the replication of the RNA. Genes for the movement protein (C) and the coat protein (D) are located near the other end of the molecule. The arrow indicates the direction of transcription. (Courtesy R. Ford). Click image to see a larger view.
The host: TMV replicates in a number of plants that are referred to as hosts. Although the virus is named for the disease it causes in tobacco, TMV can infect a wide diversity of plants from more than 9 families and over 150 genera. In several crops of economic importance (e.g., tomato), the virus causes serious losses and is a concern to growers, but in others, such as grape, infected plants show no symptoms, and the disease causes no loss of yield. Although scientists know a great deal about the virus and the diseases it causes, it still can be a concern to growers.
Hosts must allow the virus to multiply or replicate within a cell and then move to adjacent cells. If the virus spreads throughout the plant, the host is said to exhibit a systemic response. For example, in susceptible varieties of tobacco and tomato (Figure 4), the virus can be found in the roots, the stems, and the newest, noninoculated leaves. Because the virus is present in high numbers in the plants, systemic hosts are often used to increase the virus, producing large amounts of virus for study.
|Figure 4. Tomato plants systemically infected with Tobacco mosaic virus. The symptoms typically include small, curled, and puckered leaves that have light and dark green mottling, stunted plants, and a reduction of the size and number of fruit produced. (Courtesy APS). Click image to see a larger view.
In other plants, the virus remains localized near the point where it first entered the leaf. In these so-called local lesion hosts (Figure 5), the infected tissues may die, producing spots called necrotic local lesions. In other types of local lesion hosts, the tissues are not killed but chloroplasts are affected and the leaves turn yellow, producing chlorotic local lesions.
|Figure 5. Pinto bean leaves with necrotic local lesions caused
by Tobacco mosaic virus (left) [far view]. The dark brown spots on the leaves correspond to the location where the virus entered the leaf and caused the surrounding tissue to die (right) [close up]. The white irregularly-shaped areas on the leaf are typical of leaf damage caused by rubbing too hard during inoculation. (Courtesy R. Ford). Click image to see a larger view.
Variations within the virus: TMV consists of a number of strains that infect different plant species or varieties or that cause different symptoms in those plants. The common strain of TMV is found worldwide and is well characterized.
Disruption of leaf tissue: The macroscopic changes observed in infected plants are accompanied by microscopic changes. Within the mesophyll of the leaf, the normal two-layered arrangement of palisade and spongy mesophyll cells is less distinct in TMV-infected leaves than in healthy ones (Figure 6). The cells also become round and contain fewer chloroplasts. The lack of chloroplasts contributes to the lighter color or chlorosis of the leaves. This disruption of leaf tissue and cell structure also leads to a decreased rate of photosynthesis and, consequently, a reduction of sugars produced in the leaves.
|Figure 6. A comparison of healthy and Tobacco mosaic virus-infected leaf tissue. A. Healthy tissue, and B. Tobacco mosaic virus-infected tissue.(Courtesy R. Ford)
Transmission of the virus: TMV is easily transmitted between plants. The plant's epidermis, however, must be slightly wounded to allow the virus to penetrate the outer waxy layer (the cuticle) of the leaf and to reach the cells where replication occurs. Simply rubbing the leaf can cause the leaf hairs (trichomes) to break, creating wounds where TMV can enter the leaf. This ease of transmission explains why the disease can spread rapidly through contact with virus-infested tools or hands in crops like tobacco or tomato that require handling during the growing process.