A short history of plant virology. III. The thirties.

The Thirties testified on the outstanding development of plant virology: the new discoveries formalized the concept of virus on a physicochemical background. Plant viruses, which had received their own taxonomical position at the end of the Twenties, were no longer considered as simple "infective pathogens" as their size, shape and chemical nature were determined, particularly for one of them--tobacco mosaic virus (TMV). This paramount contribution was achieved as a consequence of a functional interaction between biology on one side, and chemistry and physics on the other side, from the development of which molecular biology was born. The chemical characterization of TMV developed from the first determination of nitrogen presence in purified virus, performed by Carl Vinson, through the identification of TMV as Wendell Stanley's infective, autoreplicative protein macromolecule, to the final discovery of its nucleoprotein nature by the British group of Frederick Bawden. Thorough analytical techniques--in particular electron microscopy--led to disclose the exact shape and size of TMV particle. These discoveries, that opened a new era of virology, were corroborated by new knowledge that, although less explosive, can be considered of great importance for the development of plant virology. The methodologies to estimate viral activity; the study of the relationships between viruses and insect vectors; the studies on virus spread within plants; the identification of non-sterile type of resistance and of correlation between single plant genes and viral pathogenesis benefited plant virology of a set of knowledge that, together with the discoveries on the physico-chemical properties of TMV, raised plant virology from a secondary branch of plant pathology to a new independent science by itself.     

The discovery of the chemical nature of tobacco mosaic virus.

The path to arrive at the elucidation of the chemical nature of plant viruses was greatly facilitated by the availability of Tobacco mosaic virus (TMV) as biological tool. The first hypothesis on the chemical nature of TMV was advanced in 1899 by the American Albert Wood, who suggested an enzyme nature. This hypothesis, severely questioned by Harry Hallard in 1915, was re-proposed by several virologists. In 1926, the American Maurice Mulvania concluded that the virus might be a protein with the biological characteristics of an autocatalytic enzyme. Before arriving at the experimental evidence it was necessary to resolve two questions: the estimation of virus infectivity in quantitative terms, performed by Francis Holmes in 1928, and the purification of the virus, performed by Carl George Vinson between 1927 and 1934. Vinson gave a conclusive contribution to solve the question of the chemical nature of TMV by settling the protocol of TMV purification. He put forward the hypothesis of the protein nature in the early 1930s but had not the required firm belief to gave the final experimental evidence of it. Who first arrived at the experimental evidence of the protein nature of the virus was the American Wendell Meredith Stanley, in 1935. His celebrated work, a classic of the fundamental Virology, was followed by several papers in which this result was firmly reaffirmed. The heuristic value of Stanley's discovery held out a year: the decisive evidence of the actual chemical nature of TMV was offered in the late 1936 by an English group under the leadership of Frederick Charles Bawden. In their short paper, Bawden and co-operators demonstrated that TMV had a ribonucleoprotein nature, a result that was confirmed in the following years for several TMV strains and other viruses. Stanley and his group did accept this result only after a year of reticence and contradictions. The conversion to the ribonucleoprotein nature raised a dignified protest by Bawden and the sarcasm of his closest co-operator, Norman Wingate Pirie, because Stanley proved to be very reluctant to recognize the merit of the English group. The world of Virology continues to consider Stanley as the first scientist who elucidated the actual nature of a virus, and this eminent scientist was awarded the Nobel Prize for Chemistry, in 1946. By examining the papers Stanley published from 1937 to 1945, one can however find proof of his ambiguity, a fact that justifies the bitterness of Bawden and the sarcastic comments of Pirie.     

Tobacco mosaic virus RNA as genetic determinant: genesis of a discovery.

It is generally held that the American geneticists Alfred Hershey and Martha Chase were the first to elucidate, in 1952, the genetic functions of phage DNA. The discovery of the genetic functions of RNA in a plant virus (Tobacco mosaic virus, TMV) is commonly attributed to the American plant virologist Heinz Fraenkel-Conrat, and to the Germans Alfred Gierer and Gerhard Schramm, who came to the same conclusion independently in 1956. In reality, the first understandings dated back to about 1940, when several scientists discovered that TMV infectivity was closely related to the presence of undamaged RNA in the virus particles. A very important but underestimated contribution came from the English group of Roy Markham, Kenneth Smith and Richard Matthews in 1948. This group purified and characterized an isometric plant virus, Turnip yellow mosaic virus, and first showed that virus infectivity depended on the presence of the RNA, concluding that nucleic acid was essential for virus multiplication. This finding was confirmed by the same group one year later but it laid neglected. After a five year period, in which several groups attempted to solve the question of the function of TMV RNA, the American electron microscopist Roger Hart offered, in 1955, further direct evidence which correlated RNA to TMV infectivity. One year later, three research groups (Fraenkel-Conrat; Gierer and Schramm; Max Lauffer, David Trkule and Anne Buzzell) obtained evidence that put an end to the question, which was (and is) fundamental to molecular Genetics because it demonstrated that RNA can function independently of DNA.     

A history of plant virology. Mendelian genetics and resistance of plants to viruses.

Virology was borne at the end of the nineteenth century, some years before the re-discovery of the so-called "Mendel's Laws". The rapid development of genetics was helpful to horticulturists and plant pathologists to produce hybrids of important cropping species resistant to several virus diseases. The concepts of Mendelian genetics were applied to plant virology by Francis Oliver Holmes, an American scientist who must be considered a pioneer in several fields of modern plant virology. During the Thirties, Holmes studied in particular the hypersensitive response of solanaceous plants to TMV and discovered the N dominant gene of tobacco hypersensitive to this virus. After the Second World War, the theoretic and practical support given by geneticists assisted plant virologists in better understanding the mechanism of inheritance of the character "resistance". The major problems posed by breeding for plant resistance were detected and critically discussed in several reviews published between the Fifties and the Sixties. These results, together with the discovery of the genetic functions of RNA virus raised interest on the possible relations between viral and plant genes. This fundamental subject saw the entry into the virological scene of molecular genetics, and in 1970 the Russian virologist Joseph Atabekov introduced host specificity to viruses as a central point of plant virology. From the mid 1980s, this point attracted the interest of several virologists, and many results led to several theoretic models of genetic interactions between plant and virus products. In the last fifteen years, the introduction of transgenic plants has given a remarkable contribution to the question of host specificity, which, however, still awaits a general explanation.     

Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation

Early in the development of molecular biology, TMV RNA was widely used as a mRNA [corrected] that could be purified easily, and it contributed much to research on protein synthesis. Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non-overlapping. In 1982, Goelet et al. determined the complete TMV RNA base sequence of 6395 nucleotides. The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome. Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs. The initiation site for assembly of TMV particles was also determined. However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase. As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology. To recover from this setback, a gene-manipulation system was needed for RNA viruses. In 1986, two such systems were developed for TMV, using full-length cDNA clones, by Dawson's group and by Okada's group. Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome. Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell-to-cell movement function. Many other plant viruses have since been found to encode comparable 'movement proteins'. TMV thus became the first plant virus for which structures and functions were known for all its genes. At the birth of molecular plant pathology, TMV became a leader again. TMV has also played pioneering roles in many other fields. TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro. It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene. Also, in the field of plant biotechnology, TMV vectors are among the most promising. Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology.     

Making a Virus Visible: Francis O. Holmes and a Biological Assay for Tobacco mosaic virus

In the early twentieth century, viruses had yet to be defined in a material way. Instead, they were known better by what they were not – not bacteria, not culturable, and not visible with a light microscope. As with the ill-defined “gene” of genetics, viruses were microbes whose nature had not been revealed. Some clarity arrived in 1929 when Francis O. Holmes, a scientist at the Boyce Thompson Institute for Plant Research (Yonkers, NY) reported that Tobacco mosaic virus (TMV) could produce local necrotic lesions on tobacco plants and that these lesions were in proportion to dilutions of the inoculum. Holmes’ method, the local lesion assay, provided the first evidence that viruses were discrete infectious particles, thus setting the stage for physicochemical studies of plant viruses. In a field where there are few eponymous methods or diseases, Holmes’ assay continues to be a useful tool for the study of plant viruses. TMV was a success because the local lesion assay “made the virus visible” and standardized the work of virology towards determining the nature of the virus.     

Genetics and virology: two interdisciplinary branches of biology.

Genetics has a tradition that dates back to the Ancient Greeks. It developed, between insight and contradiction, from the post-Renaissance to the mid-1800s, when Mendel and Darwin gave it the first experimental and conceptual bases. From 1910, genetics became a true experimental discipline of Biology thanks to the work of Morgan's group. On the contrary, virology is a relatively young discipline which had origin only after the success of the "germ theory" of Pasteur and Koch, by the hypothesis of the contagium vivum fluidum of Beijerinck, in 1898. In spite of their historical difference, the modern development of the two disciplines had a close connection. In 1922, the geneticist Muller first compared the bacteriophage to the gene and, in 1923, the (phyto)physiologists Benjamin Duggar and Joanne Karrer Armstrong suggested the analogy between gene and Tobacco mosaic virus (TMV). Knowledge on the biochemical nature of gene and virus developed in the early 1940s when the biochemists began to suspect that the nucleic acids might be the genetical determinants for both the bionts. Avery and co-workers discovered in 1944 that DNA was the principle of the transmission of hereditary characters in bacteria and, in 1948, a little group of English (phyto)virologists (Markham, Matthews and Smith) discovered that the RNA of a plant virus (Turnip yellow mosaic virus) was directly involved in virus replication. The fundamental significance of the two discoveries was not gathered by geneticists and virologists, even because the respective groups did not gave the necessary emphasis to their results. Thus, the discovery of the role of the nucleic acids in virus replication is historically attributed to Hershey and Chase for DNA phage, and to Fraenkel-Conrat and the German virologists Gierer and Schramm for plant viruses.     

Milestones in the research on tobacco mosaic virus

Beijerinck's (1898) recognition that the cause of tobacco mosaic disease was a novel kind of pathogen became the breakthrough which eventually led to the establishment of virology as a science. Research on this agent, tobacco mosaic virus (TMV), has continued to be at the forefront of virology for the past century. After an initial phase, in which numerous biological properties of TMV were discovered, its particles were the first shown to consist of RNA and protein, and X-ray diffraction analysis of their structure was the first of a helical nucleoprotein. In the molecular biological phase of research, TMV RNA was the first plant virus genome to be sequenced completely, its genes were found to be expressed by cotranslational particle disassembly and the use of subgenomic mRNA, and the mechanism of assembly of progeny particles from their separate parts was discovered. Molecular genetical and cell biological techniques were then used to clarify the roles and modes of action of the TMV non-structural proteins: the 126 kDa and 183 kDa replicase components and the 30 kDa cell-to-cell movement protein. Three different TMV genes were found to act as avirulence genes, eliciting hypersensitive responses controlled by specific, but different, plant genes. One of these (the N gene) was the first plant gene controlling virus resistance to be isolated and sequenced. In the biotechnological sphere, TMV has found several applications: as the first source of transgene sequences conferring virus resistance, in vaccines consisting of TMV particles genetically engineered to carry foreign epitopes, and in systems for expressing foreign genes. TMV owes much of its popularity as a research mode to the great stability and high yield of its particles. Although modern methods have much decreased the need for such properties, and TMV may have a less dominant role in the future, it continues to occupy a prominent position in both fundamental and applied research.     

Tobacco mosaic virus and the study of early events in virus infections

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.     

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.     

Studies on the Resistance Mediated by Three Mutants of TMV 54K Protein Gene

Three deletion mutants of tobacco mosaic virus (TMV) 54-kD putative replicase gene (54K) were obtained by PCR, and cloned into plant expression vector p208, then transformed into Nicotiana tabacum L. cv. SR1 by Agrobacterium tumefaciens (Smith et Townsend) Conn Ti plasmid-mediated transformation. All the transgenic plants with the N-terminal deletion mutant, the C-terminal deletion mutant and the only 261 nucleotides region from the central part of the 54K ORF showed significant resistance against TMV.      

Studies on Petunia hybrida Transformed with Flower-meristem-identity Gene AP1

Three deletion mutants of tobacco mosaic virus (TMV) 54-kD putative replicase gene (54K) were obtained by PCR, and cloned into plant expression vector p208, then transformed into Nicotiana tabacum L. cv. SR1 by Agrobacterium tumefaciens (Smith et Townsend) Conn Ti plasmid-mediated transformation. All the transgenic plants with the N-terminal deletion mutant, the C-terminal deletion mutant and the only 261 nucleotides region from the central part of the 54K ORF showed significant resistance against TMV.     

Model System for the Adsorption of Tobacco Mosaic Virus

Materials which can adsorb tobacco mosaic virus (TMV) were isolated from tobacco leaves and studied for applicability as a model system for TMV adsorption. Leaves were homogenized and fractionated by sucrose density gradient centrifugation. One fraction adsorbed TMV in the presence of polyornithine. Deduced from its sensitivity to trypsin and detergent as well as from its manner of isolation, the material responsible for adsorption of TMV seemed to be cytoplasmic membrane. Membrane derived from light particulate, as well as cytoplasmic membrane, seemed to be capable of adsorbing TMV. Shorter rods obtained by sodium dodecyl sulfate or sonic treatment of TMV could adsorb to membrane as efficiently as TMV. Viral protein subunit could not adsorb whereas helical rods made of viral protein aggregates could. A two-step nature of the adsorption of TMV was suggested: a salt-sensitive and a subsequent salt-resistant steps. In the first step, ionic bonding plays a main role in the combination between TMV and membrane. Adsorption of ¹⁴C-labeled TMV was inhibited by an excess amount of non-labeled TMV or cucumber green mottle mosaic virus but not by potato virus X or rice dwarf virus, suggesting the specific nature of adsorption. In contrast to the observed specificity on the part of virus, a membrane fraction isolated from various plants, including non-hosts for TMV, could adsorb TMV. This may imply that adsorption and injection are not the determinant of host specificity in plant viral infection.     

肿瘤病毒的发现历程及在肿瘤发生机制中的研究意义

1911年Peyton Rous发现禽肉瘤病毒,从而建立了肿瘤病毒学这一学科领域。20世纪30年代,Richard发现哺乳动物肿瘤病毒,60年代发现第一个人类肿瘤病毒--EB病毒,随后相继鉴定出乙型肝炎病毒(HBV)和乳头状瘤病毒(HPV)。肿瘤病毒的深入研究带动了癌基因概念的确立和抑癌基因功能的发现,促进癌症疫苗的研究,后者可以抑制病毒的传染性并降低肿瘤的发病率。20世纪80-90年代发现了人T细胞白血病Ⅰ型病毒(HTLV-1),丙型肝炎病毒(HVC)及卡波西肉瘤病毒(KSHV)。目前已知6种病毒(EBV、HBV、HPV、HTLV-1、HCV、KSHV)引起世界范围10%-15%的癌症,因此,病毒不仅是许多人类癌症的病原体,还可以作为揭示人类恶性肿瘤发病机制的研究工具。     

The origin of phage virology.

The history of bacteriophage (phage) had its start in 1915, when Twort isolated an unusual filterable and infectious agent from excrete of patients struck by diarrhoea; this discovery was followed by an analogous, and probably independent, finding of d'Hérelle in 1917. For several years phage research made scant progress but great attention was paid to the question of phage nature, which saw the contrast between d'Hérelle and Bordet's views (living against chemical nature, respectively). This situation changed with the independent discovery of lysogeny, in 1925, thanks to Bordet and Bail: this phenomenon was considered of genetical origin, a view that Wollman interpreted by assimilating the properties of phage to those of gene (according to a previous idea of Muller). In the 1930s, Burnet's work opened a new era by demonstrating the occurrence of several species of phages and their antigenic property. In the same period, the physical and chemical characteristics of these viruses were disclosed thanks, in particular, to the work of Schlesinger, who first demonstrated that a virus (phage) was constituted of nucleoproteins. The peculiarity of phage was finally shown after the invention of electron microscope: H. Ruska, in 1940, and Anderson and Luria in the next years, obtained the first images of tailed phages, a finding that strongly helped the investigation on the first steps of the infection process. The decisive impulse to phage virology came from Delbrück, a physicist who entered biology giving it a new arrangement. The so-called "phage group" assembled brilliant minds (Luria, Hershey and Delbrück himself, and later a dozen of other scientists): this group faced three fundamental questions of phage virology, i.e., the mechanisms of attack, multiplication and lysis. In ten years' time, phage virology became an integrant part of molecular biology, also thanks to the discovery of the genetical properties of DNA: in such scientific context, Delbrück, Luria and Hershey's works emerged for the absolute excellence of their results, which led such scientists to Nobel prize. Lysogeny was however neglected by the phage group: this singular property shared by bacteria and phages was instead investigated by Lwoff's group, in Paris, and explained in its fundamental features during the 1950s. The "phage's saga" has gone on being an important division of molecular biology till today, and its history is far from being over.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

Chimeras between Oilseed rape mosaic virus and Tobacco mosaic virus highlight the relevant role of the tobamoviral RdRp as pathogenicity determinant in several hosts

Oilseed rape mosaic virus (ORMV) is a tobamovirus taxonomically distinct from the type member of the genus, Tobacco mosaic virus (TMV). Both viruses display a specific host range, although they share certain hosts, such as Arabidopsis thaliana , Nicotiana benthamiana and N. tabacum , on which they induce different symptoms. Using a gain-of-symptom approach, we generated chimeric viruses, starting from a TMV infectious clone, over which different regions of ORMV were exchanged with their corresponding regions in the TMV genome. This approach allowed the association of pathogenicity determinants to certain genes within the ORMV genome. A general trend was observed associating the viral origin of the RNA-dependent RNA-polymerase ( RdRp ) gene and the gain of symptoms. In A. thaliana and N. benthamiana , chimeric viruses were unable to reproduce the symptoms induced by the parental viruses, leading to disease states which could be described as intermediate, and variable in some cases. In contrast, a hypersensitive reaction caused by both of these viruses on N -gene-bearing tobaccos could be found in resistance reactions to all chimeric viruses, suggesting that the avirulence determinant maps similarly in both viruses. A systemic necrotic spotting typical of non- N -gene tobaccos infected with ORMV was associated with the polymerase domain of RdRp . To our knowledge, this is the first time that this controversial portion of the tobamovirus genome has been identified directly as a pathogenicity determinant. None of the reactions of the chimeric viruses could be correlated with increases or decreases in virus titres in the infections.     

Mutant plant viruses with cell binding motifs provide differential adhesion strengths and morphologies

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths.