Encyclopedia of Virology (eBook)

FOREWORD:
TMV – 100 YEARS OF CONTRIBUTIONS TO VIROLOGY

Tobacco mosaic virus (TMV) has played a prominent role in the development of the concept of viruses as pathological agents and in unraveling the composition and structure of these unique agents. It also is the virus with which many concepts and phenomena unique to plant virology have been discovered. The mosaic disease which afflicted tobacco was known in Europe since tobacco was introduced there in the 17th century. It causes light and dark green areas on infected plants, accompanied by considerable stunting and loss of yield. The first person to transmit it experimentally was Adolph Mayer who in 1886 reported that when he took juice from diseased plants, it was infectious to healthy plants; he also gave the disease its name. The capacity to transmit the disease experimentally facilitated the studies which ultimately resulted in depicting the new class of infectious agents, the viruses.

Attribution for the discovery of the virus concept is in dispute, ascribed by some to Dmitrii Ivanowski in 1892, but by others, to Martinus Beijerinck in 1898. The basis for the dispute lies in the interpretation of the results both workers obtained when tobacco sap was passed through a porcelain, bacteria-retaining filter; the agent had retained its infectivity. Ivanowski found it difficult to accept that the agent was something new, even after he was aware of Beijerinck’s conclusions. Ivanowski was concerned that the filter might have had a crack, or that some small spore of the bacterial causal organism had passed through. Beijerinck, on the other hand (1898) concluded that he had a unique pathogen, which he termed a ‘contagium vivum fluidum’ – a contagious living fluid. Animal virologists will also argue that early work on foot-and-mouth disease also should be considered for attribution for the virus concept (see foreword by Fred Brown in this volume).

In the years before the agent was purified and characterized, several findings were made using TMV that had important ramifications for plant virology. In 1929, McKinney discovered the phenomenon of ‘cross protection’, i.e., that infection of a plant with a mild strain of a virus would protect the plant from disease when inoculated subsequently with a severe form of that virus, but would not protect it against unrelated viruses. This phenomenon was used for many years to determine virus relationships, and in a few cases it was used to protect crops against disease. Additionally, in 1929 Holmes developed a quantitative bioassay for the virus using host plants that developed necrotic local lesions in response to infection, thereby potentiating experiments where virus quantification was important.

Prior to the ultimate isolation of the infectious agent in the 1930s, a number of studies gave clues to its nature and pointed the direction for its isolation. By injecting sap from infected plants into rabbits, Purdy (1929) demonstrated that the agent was immunogenic; others found that the infectious agent could be precipitated from tobacco sap with protein precipitants, both findings hinting at a proteinaceous agent. Further, birefringence experiments suggested it was elongate. Purification was being actively pursued in the United States by several groups, and by scientists in Australia and England. Wendell Stanley, then at the Rockefeller Institute in Princeton, New Jersey, was the first to publish that he had purified the virus (1935), and he received the principal recognition, most notably the Nobel Prize in 1946. However, he incorrectly thought the virus to be composed of only protein, missing the fact that it contained both phosphorus and carbohydrate. Bawden and Pirie, who were one step behind Stanley in the purification, set the record straight in 1936, by showing that the virus contained RNA.

TMV was the first virus seen in the electron microscope (1939) and in 1941 it was the first to have its structure revealed by X-ray crystallographic analysis. Further investigations in the 1950s revealed the position of the RNA in the particle, and defined its structure.

The 1950s saw an intense rivalry between workers in Tubingen, Germany (Schramm, Melchers, Gierer, Wittmann and others) and the group in Berkeley directed by Fraenkel-Conrat at the Virus Laboratory established by Wendell Stanley. Many of the findings outlined here were made almost simultaneously in both laboratories, although partisans might not agree with such attribution. The work was greatly facilitated because TMV, unlike any other virus known at the time, was easy to purify, and because of the large quantities which could be obtained. (In my own laboratory we isolated 60 grams of highly purified virus for one of our studies.) Also, TMV – at least the common ‘strain’ – is very stable, and will maintain its infectivity for decades at refrigerated temperatures if a little chloroform is added to inhibit growth of microorganisms.

In the 1950s both groups discovered that the RNA of the virus was infectious, that the virus could be reconstituted from isolated coat protein and RNA, determined the sequence of amino acids in the coat protein, and found that chemical agents could mutate the virus. The latter studies were particularly meaningful in the confirmation of the universality of the genetic code, in that directed mutational changes induced in the RNA led to predictable changes in the viral coat protein. Additionally, mixed reconstitution experiments, utilizing proteins from one strain and RNA from another, demonstrated conclusively that the RNA was indeed the genetic material of the virus, as some had questioned that the infectivity of ‘naked’ RNA was really a reflection of a small amount of a protein contaminant. These studies demonstrated conclusively that the coat protein was coded for by the RNA component of the mixed reconstituted virus.

In 1969 Takebe and Otsuki published a paper which revolutionized plant virology when they demonstrated that cell wall-free protoplasts isolated from tobacco leaves could be infected by TMV and that TMV would replicate in those cells. Thus, plant virologists now had a system to enable single cycle analysis of virus replication, previously not possible in inoculated leaf tissues involving only a few initial infections, followed by subsequent sequential replication in surrounding layers of cells.

In 1970 my colleague V. Hari and I ascertained that TMV-infected tissues contained at least four viral proteins. Subsequent events showed these to be the 126-kDa and 183-kDa proteins of the replicase complex, the 30-kDa movement protein, and the 17.6-kDa coat protein. There is also an open reading frame for a 54-kDa protein within the read-through portion of the 126-kDa replicase protein, coincident with sequences in the 183-kDa protein. This protein has not been detected in diseased tissues, however.

Plant viruses are now known to potentiate their passage from cell-to-cell in their hosts. The 30-kDa protein of TMV was shown to be involved in this process, deduced from studies with a temperature sensitive mutant of the related tomato mosaic virus, which was defective in cell-to-cell movement. In 1987 the movement protein was shown to modify the size exclusion limit of plant plasmodesmata to allow for cell-to-cell movement, by some elegant microinjection studies from the laboratories of William Lucas and Roger Beachy. Movement proteins have now been found in most plant viruses in which they have been sought, and have been shown to have RNA binding properties.

The concept that translation of some proteins in many RNA viruses is controlled by the production of subgenomic mRNAs was first demonstrated with TMV. Virus-infected tissues were shown to contain small, virus related RNAs, and in vitro translation demonstrated their mRNA capacities.

Other phenomena of particular significance to plant virology were the detailed analysis of how the virus assembles in vivo, and the phenomenon of co-translational disassembly in which it was demonstrated that cytoplasmic ribosomes remove the coat protein from the virion, thus exposing the RNA, which is also being translated during the process to synthesize the replicase subunits. TMV was the first plant virus to be completely sequenced (1982), and was the first virus to be shown to have at least one ubiquitinated subunit in the virion. In 1996, Barbara Baker reported the first isolation of a plant gene conferring resistance to a virus. This was the N gene, utilized in the aforementioned bioassay developed by Holmes in 1929.

The important discoveries in which transformation of plants with viral sequences to induce resistance or tolerance to disease were all pioneered with TMV. The widely-adopted coat protein-mediated protection concept was developed in 1983-84 in the laboratory of Roger Beachy, then at Washington University, in collaboration with scientists at the Monsanto Company who had developed technology for plant transformation. They found that the gene for the TMV coat protein, when transformed into tobacco plants gave significant delay of symptom development, and in some cases led to complete resistance. This concept has now been widely applied to at least 30 different viruses, representing at least 15 genera. Squash plants have been marketed which are resistant to several viruses, and currently there are plantings of papaya trees in Hawaii resistant to the devastating papaya ringspot virus.

The use of replicase genes for resistance was also first shown with TMV in my laboratory in work started in 1988. We found that a portion of the 183-kDa replicase gene gave near immunity to TMV disease. This phenomenon, known as...