Viruses!

by Katrina Sahawneh

After weeks of working with stubby Arabidopsis plants, it was exciting to walk into the lab and see a table full of tall leafy plants that we soon found out were Tobacco plants, or more formally Nicotiana tabacum.

IMG_1763

Figure 1: Nicotiana tabacum plants waiting to be infected.

Even more exciting, to me at least, was that we would be infecting these plants with Tobacco mosaic virus (TMV). Previously I have done a lot of work with bacteriophage, viruses that infect bacteria, so I loved getting to work with viruses again, but on a different host. Tobacco mosaic virus is also the first virus ever to be discovered and is what first opened the world up to the idea that there were tiny, nonliving particles that could cause diseases.

So what exactly are viruses? I’m sure all of you know the name, but a virus is a nucleic acid (like RNA or DNA) surrounded by a protein coat that is able to organize its own replication only in suitable host cells. There are over 400 plant viruses, and although there are some of every type of virus, they are primarily single stranded RNA viruses. The tobacco mosaic virus is a small, 6400 nucleotide long, single-stranded RNA virus that contains 3 genes, a movement protein, a coat protein, and a replicase. The replicase also contains a leaky stop codon so that sometimes the whole  length is translated, but other times it stops the protein short so that two versions of the replicase protein are produced. Both of these versions, as well as the other two proteins, are necessary for the virus to be able to replicate itself. What really blew my mind was how small this virus’s genome was. Bacteriophage have comparatively large genomes of about 60,000 to 120,000 base pairs, and contain many different genes, many of whose function is unknown.

images

Figure 2: Electron Microscope image of the Tobacco Mosaic Virus. It is 18 by 300nm.

In the lab, we got to infect two different Nicotiana tabacum cultivars. One is susceptible, and the virus is able to spread throughout the plant and cause severe symptoms. One is resistant because it carries the N resistance gene that can recognize the replicase protein from TMV. This recognition leads to cell death that can be seen as small necrotic (grayish) lesions a few days after inoculation.

To infect leaves of the tobacco plant, we put a little carborundum powder (black silicon carbide), 20 uL of TMV solution, and then rubbed it gently around to spread it out over the leaf (Figure 3). The carborundum powder was there to make tiny cuts on the leaf’s surface to aid the virus’s passage into the plant cells, but we had to be careful not to be too rough or we would inflict mechanical damage on the plant (figure 4).

Image-1

Figure 3: Corborundum powder, the added 20uL of TMV, and it spread around the leaf.

Figure 4: Someone was a little too enthusiastic when spreading the virus and severely damaged the leaf on top. The leaf on the bottom has minimal damage along the veins, as comparison.

Figure 4: Someone was a little too enthusiastic when spreading the virus and severely damaged the leaf on top. The leaf on the bottom has minimal damage along the veins, as comparison.

A week later we got our results back and at first it was a little hard to see the difference between the resistant and susceptible plants. However, upon closer examination, we noticed that the resistant plants had small, gray lesions (Figure 5), while the susceptible ones did not.

Figure 5:

Figure 5: Leaves from the TMV resistant cultivar. ‘Mock’ was infected with water as a control while ‘high’ was infected with 50ng/uL. Notice the gray lesions on ‘high’ caused by hypersensitive response and ignore the light tan mechanical damage.

It was confusing to us as to why the resistant cultivars would be showing clear symptoms, while the susceptible ones were not. Shouldn’t it be the other way around? However, the fact that we could see lesions meant that the resistant cultivars were recognizing the TMV and initiating hypersensitive response, or purposeful cell death, in order to stop the infection from spreading. The susceptible cultivar had not figured out that it was infected yet, and as the infection had not had time to spread system wide throughout the whole plant, there were not many obvious symptoms.  Figure 6 is what tobacco looks like when an infection has “gone viral” and has spread throughout the whole plant, as compared to a healthy plant in Figure 7. We confirmed that the plant was infected with TMV using a field test that was positive (Figure 8).

IMG_0721

Figure 6: Nicotiana tabacum systemically infected with TMV. You can see where the virus gets its name from as the leaves look splotchy and like a mosaic.

 

Figure 3b: healthy Nicotiana tabacum

Figure 7: healthy Nicotiana tabacum

Figure 3c: field test positive for TMV. The first band indicates the test works, and the second indicates the sample has TMV particles.

Figure 8: field test positive for TMV. The first band indicates the test works, and the second indicates the sample has TMV particles.

 

Advertisements