Brian

TMV – Plant Resistance

The Tobacco Mosaic Virus (TMV) is a plant RNA virus that is a relatively old discovery. Though it has been known of for approximately 110 years, it still causes devastation to many crops. In plants, tobacco mosaic virus leads to severe losses. It is known to infect members of nine plant families, and at least 125 individual species, including tobacco, tomato, pepper, cucumbers, and a number of ornamental flowers. There are many different strains. The main purpose of our investigation is to discover whether some plants have developed a resistance, known as coat-protein-mediated resistance, to the TMV. We would also like to discover exactly how the Tobacco Mosaic Virus functions; does it attack the nuclei of the plant cells directly; or does it attach to the chloroplast, rendering it useless?
 * Relevance:**

The goal of our experiment is to discover which plants are more susceptible to the TMV, and which have developed more of a resistance to the virus. If possible, we would also like to discover exactly how the virus works. That is, how the virus affects the cell; why it makes the cell stop working.
 * Goal:**

Most likely, there will be some plants that we have chosen that have transgenic plants with coat-protein-mediated resistance. Because of this, they will show less symptoms of the disease, and most likely live longer than the plants without this resistance. The plants without this benefit will probably begin to discolor quickly, and die off sooner.
 * General Expectations and Models:**

The variables that we are planning to control are: l The amount of TMV solution that is applied to each leaf we infect. l The conditions that the plants are under. l The amount of necessities (water, soil, etc.) that each plant is given.
 * Variables:**

The main variable that we are planning to measure is the size of the blemishes on each of the leaves that we infect.


 * Materials needed**


 * Tobacco (two pinches from one cigarette) Specify the brand of cigarette that you use for this part of the experiment mortar and pestle
 * 10 ml 0.1 M dibasic potassium phosphate buffer
 * 2 test tubes or small beakers
 * Cotton swabs
 * Emery board
 * 5 potted plants in potting soil

1. Prepare the solution for inoculation of your plants as follows: a) Place 2 pinches of tobacco from a cigarette into the mortar. Add 5 ml of buffer and grind the mixture with the pestle until it is a fine slurry. Place the slurry in a test tube or small stoppered or covered vial until it is ready for use. Label with pertinent information including the names of the people in your group. b) Prepare your control solution and place it in an appropriate labeled covered container. 2. Using the emery board, gently abrade (scrape) the surface of some of the leaves or all of your plants (experimentals and controls). Be careful: DO NOT SCRAPE THE LEAF TOO HARD OR YOU WILL DESTROY THE PLANT. Note the position of the leaves on each plant and where they have been abraded by making a sketch in your lab book and labeling appropriately. 3. Using a cotton swab, apply the tobacco slurry to the abraded leaves of the experimental plants. Set these plants in the designated area for your experimental plants. (Refer to Paint sketch.) 4. Place the plants in a well lit area for a week to 10 days. Observe your plants and record your observations every day. Make notes daily on the condition of all the leaves. Taking pictures is recommended.
 * Procedure**


 * Expectations:**

l Some plants will be affected in a more outstanding manner, and will die sooner because of the lack of resistance (coat-protein-mediated resistance) to the TMV. l The virus is most likely affecting the chloroplasts in some manner, since there is obvious discoloration in most of the infected leaves. l All plants that have a great resistance to the virus most likely have some genealogy in common; they are most likely transgenic plants that were given genes from the same plant. l As we allow the plant to grow, the blemishes will most likely grow with it; there have been signs of the disease spreading through the leaves. l The blemishes will most likely NOT cross over to other leaves. We have not seen any nearby leaves that have been infected in this manner; we believe that this disease is localized.

Appendix 1: Related Terms


 * Buffer** any substance or mixture of compounds that, added to a solution, is capable of neutralizing both acids and bases without appreciably changing the original acidity or alkalinity of the solution.

//Buffer Etymology// 1835, from obsolete verb //buff// "make a dull sound when struck," from O.Fr. //buffe// "a blow;" hence "something that absorbs a blow."

Example:


 * Capsid** The protein shell that surrounds a virus particle

=//Capsid Etymology//= //[From Latin, box.]//

Example:


 * Chlorophyll** the green coloring matter of leaves and plants, essential to the production of carbohydrates by photosynthesis

//Chlorophyll Etymology// //1819, from Fr.// chlorophyle //(1818), coined by Fr. chemists Pierre-Joseph Pelletier (1788-1842) and Joseph Bienaimé Caventou (1795-1877) from Gk.// khloros //"pale green" (see Chloe) +// phyllon //"a leaf."//

Example:


 * Chloroplasts** A plastid in the cells of green plants and green algae that contains chlorophylls and carotenoid pigments and creates glucose through photosynthesis. In plants, chloroplasts are usually disk-shaped and can reorient themselves in the cell to vary their exposure to sunlight. Chloroplasts contain the saclike membranes known as **thylakoids**, which contain the chlorophyll and are arranged in stacklike structures known as **grana.** Besides conducting photosynthesis, plant chloroplasts store starch and are involved in amino acid synthesis. Like mitochondria, chloroplasts have their own DNA that is different from the DNA in the nucleus, and chloroplasts are therefore believed to have evolved from symbiont bacteria, their DNA being a remnant of their past existence as independent organisms.

//Chlorophyll Etymology// //1819, from Fr.// chlorophyle //(1818), coined by Fr. chemists Pierre-Joseph Pelletier (1788-1842) and Joseph Bienaimé Caventou (1795-1877) from Gk.// khloros //"pale green" (see Chloe) +// phyllon //"a leaf."//

Example:

Electron Microscope Any of a class of microscopes that use electrons rather than visible light to produce magnified images, especially of objects having dimensions smaller than the wavelengths of visible light, with linear magnification approaching or exceeding a million //Electron Etymology// //coined 1891, from electric;// electronic //is 1902 in the sense of "pertaining to electrons;" 1930 as "pertaining to electronics."// Electronics //(1910) is the branch of physics and technology concerned with the penomenon of electrons in vacuums, gas, semi-conductors, etc.//

=//Microscope Etymology//= //1656, from Mod.L.// microscopium, //lit. "an instrument for viewing what is small," from Gk.// micro- //(q.v.) +// -skopion. //"means of viewing," from// skopein //"look at."// Microscopic //"of minute size" is attested from 1760s.//

Example:

Epidermis a thin layer of cells forming the outer integument of seed plants and ferns  //Epidermis Etymology// //1626, from Gk.// epidermis, //from// epi- //"on" +// derma //"skin."//

Example:

exchange and water loss
 * Guard Cell** a specialized **cell** on the undersurface of leaves for controlling gas

//Cell Etymology// //c.// //1131, "small room," from L.// cella //"small room, hut," related to L.// celare //"to hide, conceal," from PIE base// *kel- //"conceal" (cf. Skt.// cala //"hut, house, hall;" Gk.// kalia //"hut, nest,"// kalyptein //"to cover,"// koleon //"sheath,"// kelyphos //"shell, husk;" L.// cella //"store room,"// clam //"secret;" O.Ir.// cuile //"cellar,"// celim //"hide," M.Ir.// cul //"defense, shelter;" Goth.// hulistr //"covering," O.E.// heolstor //"lurking-hole, cave, covering," Goth.// huljan //"cover over,"// hulundi //"hole,"// hilms //"helmet,"// halja //"hell," O.E.// hol //"cave,"// holu //"husk, pod"). Earliest sense is for monastic rooms, then prison rooms (1722). Used in biology 17c., but not in modern sense until 1845. Meaning "small group of people working within a larger organization" is from 1925.// Cellphone //is from 1984.//

Example:

=//Infect Etymology//= c.1374, from L. //infectus,// pp. of //inficere// "to spoil, stain," lit. "to put in," from //in-// "in" + //facere// "perform" (see //factitious//). //Infection// is 1548 in sense of "communication of disease by agency of air or water" (distinguished from //contagion,// which is body-to-body communication). //Infectious// "catching, having the quality of spreading from person to person" is 1542 of diseases, 1611 of emotions, actions, etc. Example:
 * Localized Infection** an infection that is limited to a specific part of the body and has local symptoms.


 * Mesophyll** the internal tissue between the upper and lower epidermal surfaces of a plant leaf

=//Mesophyll Etymology//=

//New Latin mesophyllum, from meso meaning “middle” + Greek phyllon meaning “leaf”//
Example:

//Virus Etymology// //13////92, "venomous substance," from L.// virus //"poison, sap of plants, slimy liquid," probably from PIE base// *weis- //"to melt away, to flow," used of foul or malodorous fluids (cf. Skt.// visam //"poison,"// visah //"poisonous;" Avestan// vish- //"poison;" L.// viscum //"sticky substance, birdlime;" Gk.// ios //"poison,"// ixos //"mistletoe, birdlime; O.C.S.// višnja //"cherry;" O.Ir.// fi //"poison;" Welsh// gwy //"fluid, water,"// gwyar //"blood"). Main modern meaning "agent that causes infectious disease" first recorded 1728. The computer sense is from 1972. Adjective form// viral //was coined 1948.// Example:
 * Plant Virus** Any of various viruses that can cause plant disease (e.g., the tobacco mosaic virus). Plant viruses are economically important because many of them infect crop and ornamental plants. Numerous plant viruses are rodlike and can be extracted readily from plant tissue and crystallized. Most lack the fatty membrane found in many animal viruses, and all contain RNA. Plant viruses are transmitted in various ways, most importantly through insect bites, mainly by aphids and plant hoppers. Symptoms of virus infection include colour changes, dwarfing, and tissue distortion. The appearance of streaks of colour in certain tulips is caused by a virus.


 * Protein** Any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids. Proteins are fundamental components of all living cells and include many substances, such as enzymes, hormones, and antibodies, that are necessary for the proper functioning of an organism

//Protein Etymology// //1844, from Fr.// protéine, //coined 1838 by Du. chemist Gerhard Johan Mulder (1802-1880), perhaps on suggestion of Berzelius, from Gk.// proteios //"the first quality," from// protos //"first." Originally a theoretical substance thought to be essential to life, the modern use is from Ger.// Protein, //borrowed in Eng. 1907.// Example:

//Retrovirus Etymology 1977, earlier retravirus (1974), from re(verse) tra(nscriptase) + virus. So called because it contains reverse transcriptase, an enzyme that uses RNA instead of DNA to encode genetic information, which reverses the usual pattern. Remodeled by infl. of retro- "backwards."// Example:
 * Retrovirus** A family of viruses distinguished by three characteristics: (1) genetic information in ribonucleic acid (RNA); (2) virions possess the enzyme reverse transcriptase; and (3) virion morphology consists of two proteinaceous structures, a dense core and an envelope that surrounds the core. Some viruses outside the retrovirus family have some of these characteristics, but none has all three. Numerous retroviruses have been described; they are found in all families of vertebrates.


 * RNA** A polymeric constituent of all living cells and many viruses, consisting of a long, usually single-stranded chain of alternating phosphate and ribose units with the bases adenine, guanine, cytosine, and uracil bonded to the ribose. The structure and base sequence of RNA are determinants of protein synthesis and the transmission of genetic information. Also called //ribonucleic acid//.

//Ribo Etymology// //1931, from// ribo-, //from ribose (q.v.), the sugar component, +// nucleic

=//Nucleic Acid Etymology//= //1892, translation of Ger.// Nukleinsäure //(1889), from// Nuklein //"substance obtained from a cell nucleus."//

Example:

//Virus Etymology// //13////92, "venomous substance," from L.// virus //"poison, sap of plants, slimy liquid," probably from PIE base// *weis- //"to melt away, to flow," used of foul or malodorous fluids (cf. Skt.// visam //"poison,"// visah //"poisonous;" Avestan// vish- //"poison;" L.// viscum //"sticky substance, birdlime;" Gk.// ios //"poison,"// ixos //"mistletoe, birdlime; O.C.S.// višnja //"cherry;" O.Ir.// fi //"poison;" Welsh// gwy //"fluid, water,"// gwyar //"blood"). Main modern meaning "agent that causes infectious disease" first recorded 1728. The computer sense is from 1972. Adjective form// viral //was coined 1948.// Example:
 * RNA Virus** An RNA-containing virus; retrovirus

Solanaceae large and economically important family of herbs or shrubs or trees often strongly scented and sometimes narcotic or poisonous; includes the genera Solanum, Atropa, Brugmansia, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Petunia, Physalis, and Solandra

//No etymology found.//

Example: The Solanaceae plant family is most commonly infected with the Tobacco Mosaic Virus.

//Stoma Etymology//
//“orifice, small opening in an animal body," 1684, from Mod.L., from Gk.// stoma //(gen.// stomatos//) "mouth," from PIE base// *stom-en-//, denoting various body parts and orifices (cf. Avestan// staman- //"mouth" (of a dog), Hittite// shtamar //"mouth," M.Bret.// staffn //"mouth, jawbone," Corn.// stefenic //"palate"). Surgical sense is attested from 1937.//


 * Example:**


 * Systemic infection** is a generic term for infection caused by microorganisms in animals or plants, where the causal agent (the microbe) has spread actively or passively in the host's anatomy and is disseminated throughout several organs in different systems of the host. In the case of animals, throughout organs in the digestive, respiratory, and other systems, especially the circulatory system; in plants, throughout the xylem and/or phloem vessels, and into organs like leaves, stems, roots, tubers, fruits, et cetera.

=//Infect Etymology//= c.1374, from L. //infectus,// pp. of //inficere// "to spoil, stain," lit. "to put in," from //in-// "in" + //facere// "perform" (see //factitious//). //Infection// is 1548 in sense of "communication of disease by agency of air or water" (distinguished from //contagion,// which is body-to-body communication). //Infectious// "catching, having the quality of spreading from person to person" is 1542 of diseases, 1611 of emotions, actions, etc.

Example:


 * Tobacco mosaic virus** (TMV) an RNA virus that infects plants, especially tobacco and other members of the family Solanaceae, showing characteristic patterns (mottling and discoloration) on the leaves

//Tobacco Etymology// 1588, from Sp. //tabaco,// in part from an Arawakan (probably Taino) language of the Caribbean, said to mean "a roll of tobacco leaves" (according to Las Casas, 1552) or "a kind of pipe for smoking tobacco" (according to Oviedo, 1535). Scholars of Caribbean languages lean toward Las Casas' explanation. But Sp. //tabaco// (also It. //tabacco//) was a name of medicinal herbs from c.1410, from Arabic //tabbaq,// attested since 9c. as the name of various herbs. So the word may be a European one transferred to an American plant. Cultivation in France began 1556 with an importation of seed by Andre Thevet; introduced in Spain 1558 by Francisco Fernandes. //Tobacco Road// as a mythical place representative of rural Southern U.S. poverty is from the title of Erskine Caldwell's 1932 novel.

Example:


 * Virion** A complete viral particle, consisting of RNA or DNA surrounded by a protein shell and constituting the infective form of a virus.

//Virus Etymology// //13////92, "venomous substance," from L.// virus //"poison, sap of plants, slimy liquid," probably from PIE base// *weis- //"to melt away, to flow," used of foul or malodorous fluids (cf. Skt.// visam //"poison,"// visah //"poisonous;" Avestan// vish- //"poison;" L.// viscum //"sticky substance, birdlime;" Gk.// ios //"poison,"// ixos //"mistletoe, birdlime; O.C.S.// višnja //"cherry;" O.Ir.// fi //"poison;" Welsh// gwy //"fluid, water,"// gwyar //"blood"). Main modern meaning "agent that causes infectious disease" first recorded 1728. The computer sense is from 1972. Adjective form// viral //was coined 1948.//

Example:


 * Virus** an ultramicroscopic (20 to 300 nm in diameter), metabolically inert, infectious agent that replicates only within the cells of living hosts, mainly bacteria, plants, and animals: composed of an RNA or DNA core, a protein coat, and, in more complex types, a surrounding envelope.

//Virus Etymology// //13////92, "venomous substance," from L.// virus //"poison, sap of plants, slimy liquid," probably from PIE base// *weis- //"to melt away, to flow," used of foul or malodorous fluids (cf. Skt.// visam //"poison,"// visah //"poisonous;" Avestan// vish- //"poison;" L.// viscum //"sticky substance, birdlime;" Gk.// ios //"poison,"// ixos //"mistletoe, birdlime; O.C.S.// višnja //"cherry;" O.Ir.// fi //"poison;" Welsh// gwy //"fluid, water,"// gwyar //"blood"). Main modern meaning "agent that causes infectious disease" first recorded 1728. The computer sense is from 1972. Adjective form// viral //was coined 1948.//

Example:


 * Appendix 2:Big Ideas**

1. Differences Between Bacteria and Viruses. The differences between viruses and bacteria are numerous. Viruses are the smallest and simplest life form known. They are 10 to 100 times smaller than bacteria. The biggest difference between viruses and bacteria is that viruses must have a living host - like a plant or animal - to multiply, while most bacteria can grow on non-living surfaces. Also, unlike bacteria, which attack the body like soldiers mounting a pitched battle, viruses are guerilla fighters. They don't attack so much as infiltrate. They literally invade cells and turn the cell's genetic material from its normal function to producing the virus itself. In addition, bacteria carry all the machinery needed for their growth and multiplication, while viruses carry mainly information - for example, DNA or RNA, packaged in a protein and/or membranous coat. Viruses harness the host cell's machinery to reproduce. In a sense, viruses are not truly "living," but are essentially information (DNA or RNA) that float around until they encounter a suitable living host.

Left: Generic diagram of a bacterium. Right: Diagram of the TMV; tobacco mosaic virus. This idea is relevant to our experiment because we are infecting plants with the tobacco mosaic virus. Because we are using a virus, it may be more difficult for the plant to destroy the disease, or even to stop the disease from spreading to other parts of the plant. If we were to infect it with a bacterium instead, it would most likely be easier for the plant to fight the disease, and our experiment would last for a shorter period of time.

2. Coat-Protein-Mediated Resistance In 1986 we reported that transgenic plants that accumulate the coat protein of tobacco mosaic virus (TMV) are protected from infection by TMV, and by closely related tobamoviruses. The phenomenon is referred to as coat-protein-mediated resistance (CP-MR), and bears certain similarities to cross protection, a phenomenon described by plant pathologists early in this century. Our studies of CP-MR against TMV have demonstrated that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. However, there is little resistance to local, cell-to-cell spread of infection. CP-MR involves interaction between the transgenic CP and the CP of the challenge virus, and resistance to TMV is greater than to tobamo viruses that have CP genes more distantly related to the transgene. Using the known coordinates of the three-dimensional structure of TMV we developed mutant forms of CP that have stronger inter-subunit interactions, and confer increased levels of CP-MR compared with wild-type CP. Similarly, it is predicted that understanding the cellular and structural basis of CP-MR will lead to the development of variant CP transgenes that each can confer high levels of resistance against a range of tobamoviruses.

This diagram shows the procedure to create a transgenic plant. Certain transgenic plants accumulate a coat of the tobacco mosaic virus, and are protected from the TMV because of this phenomenon (coat-protein-mediated resistance)

This idea is relevant to our experiment because, while we observed the plants we had infected, we noticed that certain plants showed symptoms instantaneously, whereas other plants showed no signs of infection. This suggests that some plants have developed some sort of natural resistance to the tobacco mosaic virus. The above paragraph shows one of the possible reasons for this natural resistance.

3. RNA viruses An **RNA virus** is a virus which belongs to either //Group III//, //Group IV// or //Group V// of the Baltimore classification system of classifying viruses. As such, they possess ribonucleic acid (RNA) as their genetic material and do not replicate using a DNA intermediate. The nucleic acid is usually single-stranded RNA (ssRNA) but can occasionally be double-stranded RNA (dsRNA). RNA viruses generally have very high mutation rates as they lack DNA polymerases which can find and fix mistakes, and are therefore unable to conduct DNA repair of damaged genetic material. DNA viruses have considerably lower mutation rates due to the proof-reading ability of DNA polymerases within the host cell. Retroviruses integrate a DNA intermediate of their RNA genome into the host genome, and therefore have a higher chance of correcting any mistakes in their genome thanks to the action of proof-reading DNA polymerases belonging to the host cell.

This is a diagram of several different RNA viruses. The TMV is a RNA virus; a virus which only has one strand of ribonucleic acid, instead of DNA, like many other viruses.

In our experiment, the virus that we infect the plants with is an RNA virus. This means that it multiplies faster than DNA viruses. This causes the diseased plants to become infected at a quicker pace than other diseases. This also means, however, that the virus is more likely to make a mistake when it duplicates because it lacks DNA polymerases, which may cause the virus to become weaker.

Differences Between Bacteria and Viruses. http://www.cfsan.fda.gov/~dms/qa-fdb38.html

Coat-Protein-Mediated Resistance http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1692544

RNA viruses http://www.answers.com/library/Wikipedia-cid-363066056