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Lesson Plan: Tobacco mosaic virus

Symptoms of TMV-infection in susceptible plants:

Two hosts that are susceptible to TMV (tomato and pinto bean) are inoculated with the virus. Within 5 to 7 days, symptoms will appear on bean. On tomato, a systemic host, the symptoms will not appear on inoculated leaves, but on the leaves that develop after inoculation. Symptoms will appear in 1-3 weeks, depending on the variety and concentration of virus in the inoculum. These new leaves on infected tomato plants will exhibit a mottle or green and yellow color pattern and may be distorted. The plants also may be stunted (Figure 11). The virus moves throughout tissues and multiplies in most of its cells. Thus, the presence of the virus in the leaf cells can affect color (mottling) and leaf shape (often long and pointed) of leaves that were not inoculated.

Figure 11. A comparison of TMV-infected (left) and healthy (right) leaves of tomato. (Courtesy R. Ford) Click on image to see a larger view to each image.

On pinto beans, the symptoms appear only on the inoculated leaves. Small (about 1 mm), reddish-brown local lesions will appear on the inoculated leaves in a number proportional to the concentration of virus particles in the plant extract. The virus is restricted to the parenchyma cells (mesophyll) of the bean's leaf tissue, and the tissue surrounding the point of entry dies, thus creating the necrotic local lesions. The differences in symptom development between tomato and bean can be attributed to plant-virus interactions.

Procedures:

1. Plant tomatoes and pinto beans 8-10 days prior to the experiment
   Time: Planting-20 minutes; watering daily-10 minutes
   
   
2. Preparing inoculum and inoculating plants
   Time: 30 minutes (Preparing inoculum can be completed by the teacher prior to class or as a demonstration during class.)
   
   
3. Observing the development of symptoms on the plants and comparing (1) the response of the TMV-infected tomato with the mock-inoculated plants, and (2) the symptoms on tomato with those on bean.
   Time: 40 minutes, includes time for class discussion

Discussion questions:

1. Why are some plant species but not others susceptible to TMV?
   
   
2. Why do tomato and bean produce different symptoms in response to the same virus?
   
   
3. How does the virus get into the plant during inoculation?
   
   
4. What is the path of viral movement from the point of entry on the inoculated leaf to the newest leaves?
   
   
5. What might restrict the movement of the virus within the bean leaf?
   
   
6. How might students prove that the virus was present in the necrotic lesions on the bean leaf?

Changes in leaf tissue caused by infection with TMV

Differences in the anatomy of healthy and TMV-infected leaves are obvious when comparing cross sections of tissue with the compound light microscope at 100X and 400X. Regions within the mesophyll areas of TMV-infected leaves are thinner and less organized than those of healthy leaves. The palisade and spongy mesophyll layers are not well differentiated, and the mesophyll cells, particularly in the palisade layer, are more round and contain fewer chloroplasts than those in healthy tissue.

Procedures:

1. Using the compound microscope, observe cross sections of leaf tissue, one from a healthy plant and the other from a TMV-infected plant. Scan the sections first at 100X, then at 400X. (In the slides from Carolina Biological, both healthy and TMV-infected tissues are contained on the same slide.)
   Time: 5 minutes
   
   
2. Make line drawings of the healthy and the infected tissue. Label the mesophyll layers (palisade and spongy) if present, the upper and lower epidermal layers, and the vascular tissue. Describe the differences between the two.
   Time: 20 minutes (includes time for discussion)

Discussion Questions:

1. How does the virus move from cell-to-cell?
   
   
2. What is the relationship between the number of chloroplasts in the cell and the color of the leaf?
   
   
3. How might the virus disrupt the structure of the palisade and spongy mesophyll of the leaves?
   
   
4. How would the effect of TMV on the production of sugars from photosynthesis in the leaf affect the yield of tomato?

The effect of heat on the infectivity of TMV

An extract from TMV-infected tomato is treated at various temperatures then assayed on the local lesion host, pinto bean, to determine if the virus loses its ability to induce lesions during heating. This virus loses its ability to infect plants at temperatures above 50-60° C because of changes in the structure of the viral protein. When the protein structure is changed, it no longer protects the RNA from degradation. An ideal experiment would be to have the class test a range of temperatures (e.g., 25, 50, 75, and 100° C) to demonstrate the progressive loss of infectivity associated with increasing the temperature.

Procedures:

1. Plant pinto beans 8-10 days prior to the experiment. For four heat treatments, one pot of four plants will provide four half-leaves for each treatment.
   Time: Planting-15 minutes; watering daily-10 minutes
   
   
2. Prepare inoculum (can be completed by the teacher prior to class and maintained in the freezer until needed).
   Time: 15 minutes
   
   
3. Heat-treat the virus extract for 10 minutes in a water bath. (Refer to Figure 9 Materials and Methods.) Class members could be assigned different temperatures to test (e.g., 25, 50, 75 100° C). If the class time does not permit sufficient time to inoculate the leaves immediately, the samples can be frozen until the next class period.
   Time: 30 minutes
   
   
4. Inoculate of the primary leaves of pinto beans.(Refer to Figure 7 Materials and Methods)
   Time: 15 minutes
   The primary leaves of pinto bean are inoculated with the virus preparations in a half-leaf pattern; this method allows comparison of different treatments on the same plant. Several plants (4) should be inoculated similarly.
   
   
5. Observe inoculated plants and count the necrotic local lesions on the inoculated leaves for each heat treatment. Symptoms can be observed about 1 week following inoculation.
   Time: 20 minutes. (Includes time to prepare a table (Table 1) and a graph corresponding to the number of lesions versus temperature)

Table 1. Example of table to record data from heat treatment of virus extract.

Discussion Questions:

1. How does heat affect the ability of the virus to cause lesions?
   
   
2. How does heat affect the virus particle?
   
   
3. If only the RNA were present (assume the absence of enzymes that would degrade the RNA), would the heat treatments affect its ability to infect plants?

Estimation of the size of TMV using filters

An extract from TMV-infected tomato is passed through a filter (220 nm) that prevents the passage of most bacteria through the filter. Because of the virus's small size (18 nm x 300 nm), it can pass through the filter. The ability to pass through the filter is indicated by no loss of infectivity of the filtrate when tested by its ability to produce necrotic local lesions on pinto bean leaves.

Procedures:

1. Plant pinto beans 8-10 days prior to the experiment.
   Time: Planting-15 minutes; watering daily-10 minutes
   
   
2. Preparation of inoculum.
   Time: 15 minutes
   
   
3. Filtration of the virus extract through cheesecloth. (Steps 1-3 can be completed by the teacher before lab.)
   Time: 30 minutes for preparation and filtration of the virus extract through cheesecloth.
   
   
4. Centrifugation of the virus extract in a clinical centrifuge at its highest speed, then passage of the supernatant through a 220 nm filter
   Time: 20 minutes
   
   
5. Inoculation of the primary leaves of pinto bean. The primary leaves of pinto bean are inoculated in a half-leaf pattern that allows comparison of the two preparations, filtered versus non-filtered extracts, on the same plant. Several plants (4) should be inoculated similarly. Observation and counting of the number of necrotic local lesions per treatment about 1 week following inoculation.
   Time: 20 minutes

Discussion questions:

1. How does the size of a normal bacterium compare with that of a TMV particle?
   
   
2. Are there pathogens smaller than a virus?

General Questions for Class Discussions:

1. How many TMV particles will fit into one cm, lined up end-to-end?
   
   
2. Since TMV causes disease in other plant species and produces symptoms other than a mosaic, why is it given the name TMV? Could TMV be named "tomato stunting virus?"
   
   
3. Why does TMV infect only plants and not humans?
   
   
4. Why are some plants susceptible to TMV while others are not?
   
   
5. Do viruses have a life cycle much like other organisms?
   
   
6. Why are each of the four genes of the TMV genome required for infection?

Answers:

1. How many TMV particles will fit into one cm, lined up end-to-end?
   To answer this question, the student must have an understanding of the metric system. Conversion factors are listed below. The only dimension the student needs is the length of the virus, which is 300 nm.
   
   
   

   Unit	Conversion Factor
   1 cm	10 mm
   1 mm	1000 µm
   1 µm	1000 nm

   
   
   
   Using these values, about 3.3 x 10⁴ virions will fit in one cm when laid end-to-end. The equation for calculating this value is shown below:
   # particles = (10 mm/1 cm x 1000 µm/1 mm x 1000 nm/1 µm) / 300 nm
   
   
2. If TMV causes disease in other plant species and causes symptoms other than a mosaic, why is it given the name TMV? Could TMV be named "tomato stunting virus?"
   Viruses are usually named for the host and its symptoms in which the virus was first discovered. The mosaic disease of tobacco was known since the middle of the 19th century, and since tobacco was an important crop, scientists began studying it. The ability of TMV to cause a severe disease in tomato was not known until later.
   
   
3. Why does TMV infect only plants and not humans?
   The host specificity of viruses is not unlike other infectious pathogens. The bacterium Erwinia carotovora causes soft rots on carrots and several other crops, and Streptococcus pyrogenes causes strep throat in humans. The explanation of this specificity rests with the nature of the cellular machinery or the ability of the host's enzymes to complete the steps required for replication of the virus, synthesis of the genome and/or production of proteins. Simply put, human cells cannot replicate plant viruses.
   Note: Many of the activities require the students to wear gloves when handling the virus or to wash their hands after working with the virus. This step is not to prevent the virus from infecting the student, but to reduce the spread of TMV to other plants, ones that should not be receiving the virus.
   
   
4. Why are some plants susceptible to TMV while others are not?
   Plants may have different mechanisms to restrict replication and/or movement of the virus from the cell that was originally infected. First, the infected cell may not allow the virus to replicate or to move out of the cell. Cell-to-cell movement relies on the ability of the virus to move through the plasmodesmata, the channels between two cells, that are too small to allow the movement of the virus. The movement protein, a product of one of the viral genes, facilitates this movement. Another factor that can restrict viral movement results from the formation of a necrotic local lesion as on pinto bean. In this case, the virus initiates a response in the plant that kills the tissues that are infected. which limits the spread of the virus. Scientists are working on understanding mechanisms of resistance so that virus-resistant plants can be produced.
   
   
5. Do viruses have a life cycle much like other organisms?
   The life cycle of viruses begins with entry of the virus into a cell and ends with the production of a systemic disease. The steps can be summarized as follows.
   
   
   1. Entry of the virus into the cell
      
      
   2. Disassembly of the virus into its nucleic acid and protein components
      
      
   3. Expression of the viral genes. Many viruses require the production of viral proteins before replication of its nucleic acid
      
      
   4. Replication of the viral RNA
      
      
   5. Assembly of the virus particles
      
      
   6. Local cell-to-cell movement through plasmodesmata
      
      
   7. Long-distance movement within the vascular system