Milton Zaitlin is a Professor Emeritus in the Department of Plant Pathology, Cornell University, Ithaca, N.Y.
Tobacco Mosaic Virus and Its Contributions to Virology
Early studies on TMV framed the original concept of viruses; it has proved a useful model for virologists ever since
This past year marked the 100th anniversary not only of the founding of ASM, but also of the development of the virus concept, based on studies of tobacco mosaic virus (TMV). TMV played a prominent role in the development of the concept of viruses as pathological agents and in the development of knowledge of the composition of these important agents. During this past century, research on TMV contributed immensely to virology in general and to discoveries that relate specifically to plant virology.
Several special presentations helped to commemorate this latter centennial, including my ASM-sponsored lecture during the annual meeting of the American Society for Virology, held in Vancouver, British Columbia, Canada during July 1998, and a symposium, ``Tobacco Mosaic Virus: Pioneering Research for a Century,'' sponsored by the Royal Society (London), held in Edinburgh, Scotland, in August 1998. During that symposium, leading investigators (Figure 1) summarized TMV research from about 1950, and their presentations were subsequently published in a volume edited by B. D. Harrison and T. M. A. Wilson.
The Viral Concept Takes Shape
Europeans recognized tobacco mosaic disease soon after tobacco was introduced there in the 17th century. The disease is characterized by light and dark green areas--a mosaic--on plant leaves, and it leads to considerable stunting and reduced yields. Adolph Mayer, Director of the Agricultural Experiment Station in Wageningen, the Netherlands, was the first researcher to transmit the disease experimentally and to name the disease. His 1886 paper describes how he injected a homogenate from a diseased plant into the vein of a healthy plant, noting that ``. . . in nine cases out of ten one will be successful in making the healthy plant . . . heavily diseased.''
Just who conceived the viral concept is a matter of dispute. Some credit Dmitrii Ivanowski for framing the concept in 1893, whereas others, including me, insist credit goes to Martinus Beijerinck for developing it by 1898. Some animal virologists argue that early work on foot-and-mouth disease led to the development of the virus concept. Friedrich Loeffler and Paul Frosch, whose studies were published in 1898, employed a similar filter while working with foot-and-mouth disease; their studies helped establish animal virology (see Rott and Siddell, 1998).
Meanwhile, the basis for the dispute over who first realized that a virus causes TMV lies in the interpretation of early results obtained when tobacco juice was passed through a porcelain, bacteria-retaining filter. Ivanowski did not accept that the infectious agent was small enough to pass through such a filter. Instead, he argued that perhaps the filter had a crack, or that some small spore of a larger organism passed through the filter to cause the infection. Beijerinck, on the other hand, concluded that a new type of filterable pathogen is responsible for the disease. He called it a ``contagium vivum fluidum''--a contagious living fluid.
Beijerinck (Fig. 2) was a very productive chemist and soil microbiologist, publishing about 140 papers between 1877 and 1927. His work on tobacco mosaic disease represented only a small portion of his research, but it is the most lasting. He also studied a number of soil-borne diseases and was the first to isolate the symbiotic bacteria from the nodules of nitrogen-fixing plants. His other accomplishments include identifying the etiologic agents of a number of bacterial and fungal plant diseases, elucidating the nutritional requirements for numerous bacteria, describing hereditary variations among bacteria, and discovering the properties of luminous and free-living, nitrogen-fixing bacteria, as well as the properties of a number of enzymes.
While delving into an attic room at her institute, Lesley Robertson of the Delft University of Technology in the Netherlands recently reexamined several objects of Beijerinck memorabilia, including a notebook containing notes and what appears to be drawings of the porcelain filter from his TMV experiments (Fig. 3). According to Robertson, Beijerinck's note-taking habits would not satisfy current standards; he kept his notes on loose pieces of paper and then transferred them to his notebook in a semilegible hand. He also intermingled many different experiments in each notebook.
Early Studies of TMV's Biological Properties
Before TMV was purified and its chemical and physical nature thoroughly studied, several other discoveries significantly influenced plant virology. For instance, several investigators studied the effects of environmental and cultural practices on TMV disease, while others studied how various treatments alter the infectivity of TMV and other viruses.
However, a bioassay was sorely needed to conduct such studies. In 1929 Francis O. Holmes reported that extracts containing the virus produce local necrotic lesions on leaves of some species of Nicotiana (tobacco). Moreover, lesion production is proportional to the virus content of the extracts. His first experiments involved the pricking of the leaf surface with insect pins dipped in sap from infected tobacco plants. Later, viral extracts were applied along the leaf surface--a method that is still in use. Although not terrible sensitive, this was--and is--the only good bioassay for plant viruses. Modern methods of detection, such as those based on serology or PCR, are more sensitive but do not discriminate between viable and non-viable virus.
That same year, H. H. McKinney discovered that after plants are infected with a mild ``strain'' of TMV, they are subsequently protected from more severe strains. This phenomenon, known as cross-protection, has helped to establish relationships among viruses. It also has been put to practical use to control a few diseases--most notably, to control tristeza disease of citruses in Brazil.
Purification of the Virus
Before TMV was isolated during the 1930s, other studies gave clues to its nature and laid the groundwork that led to its isolation. For instance, when Purdy injected sap from TMV-infected plants into rabbits in 1929, she demonstrated that the agent is immunogenic, suggesting involvement of a protein. Other researchers discovered that agents which precipitate proteins have the same effect on the infectious agent from tobacco sap. Furthermore, by applying ``stream double refraction'' techniques to tobacco sap, Takahashi and Rawlings determined that sap from TMV-infected plants contains elongated particles not found in uninfected plants. Thus they concluded that ``. . . the virus of tobacco mosaic or some substance regularly associated with it, is composed of rod-shaped particles.''
Although several groups of researchers in Australia, England, and the United States actively tried to isolate the virus, Wendell Stanley at the Rockefeller Institute for Medical Research, then located in Princeton, N.J., was the first to do so. He published his preliminary findings in 1935, and added more detail about the isolation in 1936. His efforts required processing 4,000 kg of tobacco leaves, yielding 14 kg of ``once-precipitated globulin'' from which he crystallized the virus. He concluded the virus was an autocatalytic protein; furthermore, his chemical analyses detected no phosphorus, which erroneously suggested that the particle contained no RNA.
Fred Bawden and Norman Pirie, working at the Rothamsted Institute in England, also purified the virus and set the record straight regarding its nucleic acid and carbohydrate contents. They determined that the virus had ``. . . 0.5 percent phosphorus and 2.5 percent carbohydrate.'' They duly noted that these two constituents ``can be isolated as nucleic acid of the ribose type . . .''
Stanley was awarded the Nobel Prize for his discovery in 1946. He subsequently founded the Department of Biochemistry and the Virology Laboratory at the University of California, Berkeley in 1948 (Creager, 1996).
Physical Parameters, Electron Microscopy, and Virus Structure
The work characterizing the virus was greatly facilitated because TMV, unlike any other virus known at the time, was easy to isolate and purify. Moreover, infected tobacco plants yielded large quantities of virus. For instance, in 1985 we readily isolated 60 grams of highly purified virus from 20 kg of leaf tissue for one of our studies. Also, TMV--at least the common ``strain''--is very stable, and will maintain its infectivity for decades if kept refrigerated and if an antimicrobial agent is added to prevent microorganisms from degrading it.
Thus, TMV served as a test object and became the first virus subjected to the recently developed techniques of solution electrophoresis and analytical ultracentrifugation. In 1936 Stanley provided researchers in Uppsala, Sweden with samples of purified virus which they used to determine its ``sedimentation constant'' and its isoelectric point. TMV was also the first virus to be viewed in the electron microscope (Fig. 4, upper). Little structure other than needle-like particles is evident in such older micrographs, compared to what can be observed by modern electron microscopy (Figure 4, lower). Pioneering structural studies also were under way. X-ray crystallographers in Cambridge, U.K., determined in 1941 that the rod-shaped virus consists of many orderly, helically spaced protein molecules. Those experiments were difficult, with the preparation exposed to the X-ray beam for hundreds of hours, followed by tedious mathematical analysis. Today crystals are exposed to high-energy beams for only a few minutes or seconds, and computers do the calculations!
A decade later, X-ray crystallographers, including Don Caspar, Ken Holmes, and Rosalind Franklin, in Cambridge determined that RNA resides in the interior of the TMV particle (see Piper, 1998). A few years later, Franklin supplied some of the X-ray diffraction films that proved vital to the efforts of James Watson and Francis Crick in elucidating the double-stranded helical nature of DNA.
Intensive TMV Rivalries Emerge during the 1950s
During the 1950s, an intense rivalry arose between a group of researchers in Tubingen, Germany, whose members included Gerhard Schramm, Georg Melchers, Alfred Gierer, Karl Mundry, and Gunter and Brigitte Wittmann, and a group directed by Heinz Fraenkel-Conrat at the Virus Laboratory, which Wendell Stanley established at the University of California, Berkeley. Both groups, working with virus provided to them by Stanley, described several findings virtually simultaneously.
For example, both groups proved that TMV RNA is, by itself, infectious, reinforcing the then-new argument that RNA carries genetic information. However, some researchers then found the notion that nucleic acid carries such information unacceptable, leading to heated debates over whether protein was also present and that it, rather than the RNA, was essential for infectivity. Experiments designed to purge even traces of protein from the preparations did not assuage some of those skeptics.
However, by 1957, Fraenkel-Conrat and Bea Singer put an end to those polemics. Earlier, Fraenkel-Conrat dissociated TMV into free RNA and free coat protein. Under appropriate conditions, these two components could be reconstituted into infectious virus particles. Through such experiments, they proved that RNA carries genetic information. In all they used RNA from four different viral strains, and the reconstituted particles faithfully reflected the characteristics of the viral strains from which those RNA samples were extracted. ``The nature of the disease provoked by mixed virus preparations resembled in each case that characteristic of the virus supplying the nucleic acid,'' they stated in their research paper.
Because the TMV particle appeared to have a molecular weight of 40 x 106, determining the sequence of its proteins at first seemed a hopeless undertaking. Soon, however, investigators realized that the coat protein contains a large number of identical subunits, a fact that greatly accelerated research on its composition and sequence. Nonetheless, such efforts during the late 1950s entailed a long and laborious process.
Once again, members of both the Tubingen and Berkeley groups competed over several years in their efforts to analyze this TMV component. First they purified the coat protein, and then cleaved it into shorter polypeptides, each of which had to be purified on ion exchange chromatographic columns and/or by counter current distribution. Those polypeptides then were sequenced by manual, laborious chemical methods. To reveal their order in the protein, other proteases were used to produce additional peptides with overlapping sequences.
This sequencing effort was truly a monumental task. Both laboratories published versions of the sequence in 1960 that differed only in some minor details, and those differences were later reconciled. The 158-amino-acid TMV coat protein was the third protein to be completely sequenced, and at the time it was the largest. The two others were insulin, which contains 51 amino acids, and pancreatic ribonuclease, which consists of 124 amino acids.
Molecular Biology, Physiology, and Genomic Organization of TMV
In 1970 V. Hari and I determined that TMV-infected tissues contain at least four viral proteins, which later proved to be the 126-kDa and 183-kDa replicase, the 30-kDa movement protein, and the 17.6-kDa coat protein. The viral genome also contains an open reading frame (ORF) for 54 kDa within the readthrough portion of the 126-kDa replicase gene, coincident with sequences in the 183-kDa protein. However, the protein encoded by this ORF has never been detected in diseased plant tissues.
Plant viruses potentiate their passage from cell to cell in their hosts. Masamichi Nishiguchi and colleagues in Japan determined that the 30-kDa protein of TMV is involved in this process, based on studies involving a mutant of the tomato mosaic virus that is defective in cell-to-cell movement at specific temperatures. This protein apparently modifies the size exclusion limit of plant plasmodesmata, thereby allowing cell-to-cell movement of the viruses, according to microinjection experiments done in 1987. Many plant viruses produce similar proteins.
That subgenomic mRNA production can control translation of some proteins in many RNA viruses was first shown with TMV. Virus-infected plant tissues contain small, virus-related RNAs, which other experiments prove serve as messengers.
Engineering Plant Genes for Viral Resistance
The first genetic engineering studies to induce disease resistance or viral tolerance in plants were done with TMV. For example, during the mid 1980s, Roger Beachy, then at Washington University in St. Louis, Mo., and his collaborators at Monsanto transformed plants with genes for the TMV coat protein as a means for protecting them against disease symptoms. Once the coat protein gene was transformed into tobacco plants, it significantly delayed the development of symptoms and, in some cases, led to complete resistance.
This means for protecting plants has now been widely adapted to protect other plants against at least 30 different viruses, representing at least 15 genera. For instance, transgenic squash plants with resistance to several viruses are commercially available. Moreover, in Hawaii, plantings of transgenic papaya trees are helping growers overcome infestations of papaya ringspot virus that were devastating this crop.
My colleagues and I used TMV to establish use of replicase genes to confer resistance to viruses in plants. A decade ago, we found that introducing a portion of the 183-kDa replicase gene into the host imparts near immunity to tobacco mosaic virus. This phenomenon, known as replicase-mediated resistance, also has been applied to a number of other viruses in other genera. For instance, potato plants containing the replicase gene from potato leaf roll virus are in commercial production.
The gene encoding the TMV movement protein also was the first of its kind to be tested as a means for conferring resistance to the source virus. When plants are transformed with a mutant gene specifying a defective TMV movement protein, they become partially resistant to TMV as well as other tobamoviruses and several unrelated viruses.
Beijerinck, M. J. 1898. Concerning a contagium vivum fluidum as cause of the spot disease of tobacco leaves. Verhandelingen der Koninkyke akademie Wettenschappen te Amsterdam 65:3-21. Translation published in English as Phytopathological Classics Number 7 (1942). American Phytopathological Society Press, St. Paul, Minn.
Creager, A. N. H. 1996. Wendell Stanley's dream of a free-standing biochemistry department at the University of California, Berkeley. J. Hist. Biol. 29:331-360.
Harrison, B. D., and T. M. A. Wilson (ed.). 1999. TMV: pioneering research for a century. Trans. R. Soc. B 354:517-685.
Iwanowski, D. 1892. Concerning the mosaic disease of the tobacco plant. St. Petersb. Acad. Imp. Sci. Bull. 35:67-70. Translation published in English as Phytopathological Classics Number 7 (1942). American Phytopathological Society Press, St. Paul, Minn.
Piper, A. 1998. Light on a dark lady. Trends Biol. Sci. 23:151-154.
Rott, R., and S. Sidell. 1998. One hundred years of animal virology. J. Gen. Virology 79:2871-2874.
Scholthof, K.-B., J. G. Shaw, and M. Zaitlin (ed.). 1999. Tobacco mosaic virus: one hundred years of contributions to virology. American Phytopathological Society Press, St. Paul, Minn.
Zaitlin, M. 1998. The discovery of the causal agent of the tobacco mosaic disease, p. 105-110. In S.-D. Kung and S.-F. Yang (ed.), Discoveries in plant biology. World Scientific Publishing Co., Singapore.
October 8, 1999
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