tobacco mosaic virus experiment

A Smithsonian magazine special report

How a Few Sick Tobacco Plants Led Scientists to Unravel the Truth About Viruses

With the COVID-19 coronavirus causing a global pandemic, a look back at the scientists who figured out viruses and their relationship to disease

Theresa Machemer

Correspondent

When German pathologist Robert Koch discovered the bacterium behind tuberculosis in 1882, he included a short guide for linking microorganisms to the diseases they cause. It was a windfall for germ theory, the modern understanding that pathogens can make us sick. But it didn’t only shake up the field of medicine: Botanists took note, too.

When a blight of mosaic disease threatened European tobacco crops in the mid-1800s, plant pathologists set out to identify its root cause. For decades, only one forward-thinking botanist, Martinus Beijerinck, realized the source was neither a bacterial nor a fungal infection, but something completely different: a virus.

Today, we know that viruses can be found nearly anywhere in the air , oceans and soil . A tiny percentage of these are dangerous pathogens that cause disease, such as the current coronavirus called SARS-CoV-2 causing a worldwide pandemic. Yet the study of viruses started not in medical science, but in botany, the study of plants. Viruses are so small—and so strange—that it would take decades for scientific consensus to agree that they exist at all.

Agents of Disease

The idea that microorganisms could cause plant disease wasn’t entirely new even in the late 19th century. In the 1840s, Reverend Miles Berkeley, also a botanist, identified the fungus behind Ireland’s potato blight, despite the clergy’s notion that the devil was to blame.

In 1857, farmers in the Netherlands reported a disease threatening another economically vital crop: tobacco. The leaves began turning a mottled dark green, yellow, and grey, causing farmers to lose up to 80 percent of crops in affected fields. Massive fields of tobacco that had been planted with the same crop repeatedly were especially susceptible . Once the disease reached a farmer’s field, it spread rapidly.

“It's very easy for it to move around,” says plant virologist Karen-Beth Scholthof of Texas A&M University. “If you're in a greenhouse or your garden and you're watering with a hose and the hose touches an affected plant, you can end up damaging a plant next to it.”

In the Netherlands, plant pathologist Adolf Mayer began researching the disease in 1879 and named it the “mosaic disease of tobacco.” He tried to use Koch’s guidelines, which call for a series of germ isolations and re-infections, to find its cause. But Mayer ran into trouble. Although he showed that the sap from a sick tobacco leaf could pass the disease to a healthy leaf, he couldn’t produce a pure culture of the pathogen and couldn’t spot the culprit under a microscope.

“The tools did not exist to see a virus,” says biological anthropologist Sabrina Sholts , curator of the Smithsonian National Museum of Natural History’s Outbreak exhibit. “It was just this invisible contagion.”

When botanist Dmitri Ivanovski researched tobacco mosaic disease in Crimea beginning in 1887 , he took a different approach. He strained the sap through fine filters made of unglazed porcelain, a material with pores that were too small for bacteria to squeeze through. But when Ivanovski put the filtered sap on a healthy tobacco leaf, it turned mottled yellow with disease. Ivanovski could barely believe his data, which he published in 1892. He concluded that the disease was caused by a toxin that fit through the filter or that some bacteria had slipped through a crack.

Dutch microbiologist Beijerinck independently conducted almost the same experiments as Ivanovski, but he came to a much different conclusion. The early pathologist added to the porcelain filter experiments with a second kind of filtration system that used a gelatin called agar to prove that no microorganisms survived the first filtration. Bacteria get stuck on top of the gelatin, but the mysterious mosaic-causing pathogen diffused through it.

Beijerinck also provided evidence that the disease agent relies on growing leaves to multiply. By re-filtering the pathogen from an infected leaf and using it to cause mosaic disease on another plant, he showed that the agent could spread without diluting its disease-causing power. He proved the pathogen was growing in the leaves, but strangely, it couldn’t reproduce without them.

When he published his findings in 1898, Beijerinck called the infectious, filtered substance contagium vivum fluidum— a contagious, living fluid. As a shorthand, he reintroduced the word “virus” from the Latin for a liquid poison to refer specifically to this new kind of pathogen.

“I don't think Ivanovski really understood his results,” Scholthof says. “Beijerinck set up the experiments and trusted what he saw… The way we use ‘virus’ today, he was the first one to bring that term to us in a modern context, and I would give him credit for the beginning of virology.”

A Bold Hypothesis

Although Beijerinck incorrectly thought viruses were liquid (they are particles) his results were close to the mark. Yet his idea didn’t catch on. His suggestion of a pathogen without a cell conflicted with early germ theory and was radical for the time.

Ivanovski continued to search for a bacterial cause of tobacco mosaic disease, claiming “that the entire problem will be solved without such a bold hypothesis ” as Beijerinck’s. In the meantime, researchers grappled with the evidence at hand. In 1898, the same year as Beijerinck’s work was published, foot-and-mouth disease in cattle became the first animal illness linked to a filterable agent, or a microbe small enough to pass through a porcelain filter. In 1901, American researchers studying yellow fever in Cuba concluded that the disease carried by mosquitoes was caused by something small enough to be filterable , too.

At the time, the researchers didn’t consider their discoveries to be viruses like Beijerinck’s. The prevailing theory was that there were simply bacterial that could fit through the filter. Early review articles of invisible contagions sometimes grouped barely visible bacteria with Beijerinck’s viruses.

“In the early days, there was a lot of confusion because you couldn’t see them,” Scholthof says. Questions about whether these tiny germs were small bacteria, molecules secreted by bacteria, or something else remained unanswered into the 1920s. “Some people would probably say [the questions went on] until they could be seen with an electron microscope,” she says.

A Model Virus

In 1929, biologist Francis Holmes used the tobacco mosaic virus to develop a method proving that viruses are discrete particles mixed in the filtered sap and that they have stronger effects at higher concentrations. In 1935, chemist Wendell M. Stanley created a crystallized sample of the virus that could be visualized with X-rays, earning him a share of the 1946 Nobel Prize. (The clearest X-ray diffraction image of tobacco mosaic virus came from Rosalind Franklin, in 1955 , after her contributions to the discovery of DNA’s double helix.) The first clear, direct photographs of tobacco mosaic virus would not come until 1941 with the invention of powerful electron transmission microscopes, which revealed the pathogen’s skinny, sticklike shape.

This was a turning point in the scientific understanding of viruses because visual proof dispelled any doubt of their existence. The images showed that viruses are simple structures made of genetic material wrapped in a solid coat of protein molecules—a far cry from squishy, cellular bacteria. But Beijerinck didn’t live to see his theory validated, as he died in 1931.

“In a way, we were lucky that it was this was a disease found on tobacco,” Scholthof says. “It was an economic problem. It was easy to work with and purify. The virus itself only in it encodes five genes.” Because the virus has been a research subject for so long, it was used to develop fundamental ideas in virology. It remains a tool in plant virology today.

Mayer, Ivanovski and Beijerinck’s work didn’t stop the spread of tobacco mosaic during their lifetime; tobacco production halted entirely in the Netherlands. But their pioneering work on tobacco mosaic virus opened the door to a century of research that has revealed a diverse range of viral structures and strategies for survival.

While tobacco mosaic virus is rod-shaped and made up only of genes and protein, others, like the COVID-19 coronavirus, are round and wrapped in a fatty envelope that makes them especially susceptible to soap when you wash your hands . Advancements in the understanding of how viruses spread allowed for the eradication of smallpox and the invention of several life-saving vaccinations.

“It's only been in the last century that a lot of these amazing achievements happened, and it's happened so fast and so dramatically that we almost can't relate to what the world was like,” Sholts says. Right now, “there's a lot to be concerned about and take seriously. But I usually find what the scientists are doing to be one of the brightest elements to anything that you might look at.”

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Theresa Machemer | READ MORE

Theresa Machemer is a freelance writer based in Washington DC. Her work has also appeared in National Geographic and SciShow. Website: tkmach.com

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Article Contents

Structural biology, virus genetics and evolution, cell and molecular biology, beijerinck's legacy: biotechnology, acknowledgments.

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Tobacco Mosaic Virus: Pioneering Research for a Century

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Angela N.H. Creager, Karen-Beth G. Scholthof, Vitaly Citovsky, Herman B. Scholthof, Tobacco Mosaic Virus: Pioneering Research for a Century, The Plant Cell , Volume 11, Issue 3, March 1999, Pages 301–308, https://doi.org/10.1105/tpc.11.3.301

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One century ago, M.W. Beijerinck contended that the filterable agent of tobacco mosaic disease was neither a bacterium nor any corpuscular body, but rather that it was a contagium vivum fluidum ( Beijerinck, 1898 ). Beijerinck's contribution followed A. Mayer's path-breaking work on tobacco mosaic disease and D. Ivanowski's demonstration in 1892 that the agent of tobacco mosaic disease could pass through a filter capable of retaining bacteria ( Mayer, 1886 ; Ivanowski, 1892 ; Zaitlin, 1998 ). The claim that viruses, lacking cells, were nonetheless living, set off a scientific debate about the nature of life that animated biology for decades.

Tobacco mosaic virus (TMV), as we now know the agent that Beijerinck and others were studying, was the first virus to be identified. Perhaps because of this, research on TMV and other plant viruses has continued to be of profound significance in addressing fundamental questions about the nature of viruses in general. Indeed, TMV as a model system has been at the forefront of virology research to the present time. For example, TMV was the first virus to be chemically purified ( Stanley, 1935 ; Bawden et al., 1936 ), to be detected in an analytical ultracentrifuge and in an electrophoresis apparatus ( Eriksson-Quensel and Svedberg, 1936 ), and to be visualized in an electron microscope ( Kausche et al., 1939 ). TMV RNA was used in the first decisive experiments showing that nucleic acids carry hereditary information and that nucleic acid alone is sufficient for viral infectivity ( Fraenkel-Conrat, 1956 ; Gierer and Schramm, 1956 ). The TMV coat protein (CP) was the first virus protein to be sequenced ( Anderer et al., 1960 ; Tsugita et al., 1960 ), and TMV's particle structure was among the first to be elucidated in atomic detail ( Bloomer et al., 1978 ; Namba et al., 1989 ).

TMV's preeminence has extended into the recombinant era, when the first transgenic plants were constructed using TMV to demonstrate the concept of CP-mediated cross-protection ( Abel et al., 1986 ). TMV was also the first virus shown to encode a cell-to-cell movement protein (MP; Deom et al., 1987 ). MP binds to RNA ( Citovsky et al., 1990 ), associates with cytoskeletal elements ( Heinlein et al., 1995 ; McLean et al., 1995 ), and increases the permeability of plasmodesmata to mediate cell-to-cell movement of the virus ( Wolf et al., 1989 ; Waigmann et al., 1994 ).

Several properties of TMV have made it particularly amenable to laboratory investigation. For example, infected tobacco plants produce TMV so abundantly that inclusion bodies of crystallized virions in the infected leaves are visible under the light microscope. Moreover, TMV is not transmitted by insects, nematodes, or other vectors; it infects cells via direct contact with wounded areas on plant surfaces. Virus infection causes disease by preventing chloroplast development, resulting in stunted plants with leaves showing a characteristic mosaic pattern of light and dark green. Furthermore, TMV is remarkably stable: its in vitro longevity in infected sap is 3000 days, and purified virions kept at 5°C remain viable for at least 50 years. Virus stability derives directly from the densely packed structure of the viral particles, which consist of a single genomic RNA molecule enclosed in a cylindrical protein coat. TMV virions have a regular length of 300 nm and a width of 18 nm; these rods comprise a tight array of 2130 identical CP subunits, each containing 158 amino acids.

The TMV RNA genome is single stranded and linear, with a length of ~6400 bases. The complete TMV nucleotide sequence was first determined for the U1 strain ( Goelet et al., 1982 ), and comparisons between this sequence and the RNA sequences of other TMV strains have revealed a tightly organized genome that encodes at least three nonstructural proteins (P126, P183, and the 30-kD MP), a putative 54-kD protein of unknown function, and the CP ( Figure 1 ). Both P126 and P183 function as components of the TMV replicase ( Palukaitis and Zaitlin, 1986 ) and are translated directly from the genomic TMV RNA; P183 is produced by read through of the amber termination codon of P126 ( Pelham, 1978 ). In addition, there is a start codon within this read-through region and an open reading frame that could potentially encode the putative 54-kD protein. Translation of MP and CP occurs from the I 2 RNA and CP subgenomic (sg) RNAs, respectively ( Figure 1 ). Whereas P126, P183, and CP are continuously expressed ( Watanabe et al., 1984 ), translation of MP is transient, occurring early in the infection process ( Joshi et al., 1983 ; Watanabe et al., 1984 ).

To celebrate the first century of TMV research, scientists from around the world gathered at the Royal College of Physicians of Edinburgh, Scotland on August 7 and 8, 1998, for a symposium sponsored by the Royal Society of Edinburgh in association with The Royal Society, London, UK. The meeting was organized by Professors Bryan D. Harrison and T. Michael A. Wilson (both of the Scottish Crop Research Institute, Dundee, UK) to consider how studies on TMV have contributed to the fundamental knowledge base of biology. A wide diversity of research fields—crystallography, plant pathology, immunology, biochemistry, genetics, and evolutionary biology—was represented at the meeting, with distinguished symposium speakers describing the contributions made by their respective disciplines to our current understanding of TMV biology. The overview they provided made clear that the special status of TMV as a research object has depended on its biological characteristics as well as upon its historical status as a virus of many “firsts.”

In the remainder of this report, we describe symposium presentations focused on four central approaches to TMV research—structural biology, genetics and evolution, cell and molecular biology, and biotechnology—and we emphasize the impact that research on TMV has had among the life sciences over the course of the twentieth century.

Current Molecular Biological Conception of TMV, as Represented by a Schematic Diagram of its Genome Organization.

The genomic RNA is shown on top and the 3′ coterminal subgenomic (sg) RNAs are shown underneath. The predicted molecular weights of proteins encoded by the open reading frames (boxes) are given in kilodaltons, and the functions of individual genes are indicated (the function of the putative 54-kD protein is unknown). The asterisk denotes the amber read-through codon.

The intensive study of the structure of TMV has established it as one of the best-investigated models of macromolecular organization in biology. The classic reconstitution experiments, in which complete TMV was produced in vitro by mixing purified virus RNA and protein subunits ( Fraenkel-Conrat and Williams, 1955 ), demonstrated that the information required for assembly is present in the structural components of the virus. Subsequent studies of the self-assembly of the virus have drawn from the wealth of biophysical and biochemical data on the various stable aggregates of TMV CP, as Donald L.D. Caspar (Florida State University, Tallahassee) noted in his contribution to the symposium. By the late 1950s, M. Lauffer's physicochemical studies of the TMV CP along with Caspar's own titration studies had provided evidence that disks comprising two cylindrical layers of 17 subunits each might serve as important intermediates for the assembly of virus helices ( Lauffer et al., 1958 ; Caspar, 1960 ).

During the ensuing four decades, a great deal of progress has been made in resolving the structure and function of these 34-subunit disks, although the degree to which disks are involved in virus assembly remains controversial. P.J.G. (Jo) Butler (MRC Laboratory of Molecular Biology, Cambridge, UK) recounted the evidence amassed by him, Klug, and their coworkers showing that virus disks are essential for self-assembly. According to their model, nucleation is initiated by the binding of an internal sequence of TMV RNA to a disk, which then dislocates into a helical structure. Other dislocated disks associate with the initiation complex to form nicked helices. Over time, the protein subunits realign and anneal into an uninterrupted helical rod ( Butler, 1984 ). Butler presented kinetic evidence implicating the disks in rod elongation as well as nucleation.

Butler and Klug's assertions did not go unchallenged, however. Marc H.V. Van Regenmortel (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France) pointed out that only polar disks can form helices. The stacked disks produced by most in vitro experiments are bipolar, and thus cannot, in his view, represent intermediates in the assembly process. Recognizing that the problem of TMV self-assembly has not been entirely settled, Klug reminded the audience of A.N. Whitehead's famous dictum, “It is more important that an idea be fruitful than correct.”

The desire to ascertain the structure of complete TMV, rather than only of smaller oligomeric subunits, has required the development of innovative crystallographic methods. Gerald Stubbs (Vanderbilt University, Nashville, TN) recalled a 1971 talk on TMV structure by K.C. Holmes that inspired his efforts to push the resolution of TMV structure below 10 Å. The conventional reliance on cylindrical averaging of the fiber diffraction data, although yielding the overall architecture of the virus, had effectively obscured more detailed structural information.

Stubbs and his coworkers developed new isomorphous replacement and computational techniques to achieve atomic resolution of intact TMV, providing new structural details of both the RNA and the capsid subunits, and by 1989, they had obtained a 2.9-Å map of TMV ( Namba et al., 1989 ). Stubbs pointed out that the techniques developed to extend the resolution of TMV have been useful for investigating other complex biological structures, including filamentous bacteriophages.

As Yoshimi Okada (Teikyo University, Utsunomiya, Japan) reminded those attending the symposium, it was not until the 1950s that most biologists accepted that genes are constructed from nucleic acids. Experiments with TMV played an important role in this development by providing the first unequivocal demonstration that a viral RNA molecule—specifically the TMV RNA—was sufficient for infectivity and carried all of the information necessary for synthesis of the CP ( Fraenkel-Conrat, 1956 ; Gierer and Schramm, 1956 ).

Bea Singer (University of California, Berkeley) recounted how she and H. Fraenkel-Conrat (University of California, Berkeley) extended biochemical research of TMV genetics. They began with naturally occurring TMV strains to demonstrate that the progeny of mixed viruses (i.e., protein from one strain and RNA from another) were true-to-type for the TMV nucleic acid ( Fraenkel-Conrat and Singer, 1957 ). Fraenkel-Conrat and Singer subsequently employed the mutagen nitrous acid ( Gierer and Mundry, 1958 ) to generate novel variants of TMV that were then compared in terms of their nucleic acid content, CP composition, and disease symptoms. Due to the labile nature of the TMV RNA, these were difficult experiments. As Singer recalled, she and Fraenkel-Conrat protected the RNA from cellular RNases by adding the clay bentonite, leading their colleague C.A. Knight to remark that he “wouldn't put that mud in his stuff.”

Singer asserted that her work with Fraenkel-Conrat represented the true beginning of chemistry applied to virology. However, one might well point out that this work drew on concurrent developments in bacteriology and bacterial genetics, beginning with research performed a decade earlier at the Rockefeller Institute, where Avery and his coworkers biochemically demonstrated the “transforming principle” of Streptococcus to be DNA.

With the elucidation of the complete CP sequence in 1960 ( Anderer et al., 1960 ; Tsugita et al., 1960 ) the collection of TMV mutants provided clues used to crack the genetic code. Only the startling development of cell-free translation systems the following year by H. Matthei and M. Nirenberg provided a less laborious means to decipher this code (reviewed in Kay, 1998 ), and even then TMV mutants were used to confirm the emerging codon dictionary.

The TMV mutants shed light on other biological questions as well. As Singer also noted, almost all the mutants attributed to the nitrous acid treatment were less “fit” than was wild-type TMV, an observation suggestive of later developments in the arenas of virus diversity and evolution.

Milton Zaitlin (Cornell University, Ithaca, NY) recalled how advances in molecular genetic techniques enabled researchers in the 1970s and 1980s to construct a detailed map of the TMV genome. Indeed, a significant clue to the genetic composition of TMV RNA came from studies in Zaitlin's laboratory showing that a low molecular–weight component termed sgRNA accumulated during viral infection ( Jackson et al., 1972 ). This sgRNA was soon implicated as the mRNA that directs CP production ( Hunter et al., 1976 ). By the mid-1970s, Zaitlin's group had correctly, albeit tentatively, placed the replicase-encoding gene at the 5′ end, the CP-encoding gene at the 3′ end, and a gene necessary for viral movement in the central portion of the genome ( Beachy et al., 1976 ; see Figure 1 ). Nishiguchi, Okada, and coworkers ( Nishiguchi et al., 1978 ; Ohno et al., 1983 ) then confirmed that the 30-kD MP was encoded by TMV using TMV strain L and a temperature sensitive variant, Ls-1. Several reverse genetics studies using infectious clones, first assembled in 1986 ( Dawson et al., 1986 ; Meshi et al., 1986 ), have confirmed the gene functions assigned during these earlier studies.

The initiation of TMV infection and disassembly of the TMV virion was reviewed by John G. Shaw (University of Kentucky, Lexington). Having entered its host cell, the TMV virion must remove its CP to enable viral replication. Shaw presented one model describing how this uncoating might take place bi-directionally, proceeding both from the 5′ and the 3′ ends of the TMV genomic RNA molecule. The 5′-to-3′ uncoating reaction may be cotranslational ( Wilson, 1984 ), which would result in disassembly by a ribosome-mediated mechanism and concomitant protection of the uncoated viral RNA from cellular nucleases. Shaw suggested that the 3′-to-5′ uncoating reaction might occur in a coreplicational manner, because viral replicase mutants that are defective in 3′-to-5′ disassembly can be uncoated in plant protoplasts by adding free viral RNA with an intact replicase gene (Wu and Shaw, 1996 , 1997 ). These studies of TMV infection at the molecular level exemplify the highly efficient coordination of seemingly disparate events associated with virus replication.

Ken Buck (Imperial College of Science, Technology, and Medicine, London, UK) dissected the process of TMV replication, and he noted that although the viral proteins involved in TMV replication are well characterized, the involvement of host factors is poorly understood. On the basis of their physical association with the viral replicase proteins, Buck proposed two candidate host proteins that might be involved in TMV RNA synthesis: EF-1α, which colocalizes with the replicase complex, and a subunit of eIF-3, which copurifies with the replicase. In addition, Buck mentioned genetic approaches to dissect virus–host interactions that have led to the identification of the tom-1 and tom-2 mutants of Arabidopsis and the Tm-1 mutant of tomato, in which TMV replication is restricted.

Population genetic studies with tobamoviruses have also yielded surprising results. Although RNA viruses have the potential to vary more widely than DNA viruses ( Domingo and Holland, 1994 ), TMV provides a case of high genetic stability. Adrian J. Gibbs (Australian National University, Canberra) compared cDNA sequences of recent isolates of tobacco mild green mottle tobamovirus (TMGMV) and TMV with those derived from infected Nicotiana glauca specimens deposited in Australian herbaria since 1899. Gibbs reported that these analyses demonstrate that there has been no increase in the genetic diversity of TMGMV in Australia over the past 100 years. Moreover, the mutations observed in TMV seem to be deleterious because TMGMV has become the more dominant tobacco virus in N. glauca in that country ( Fraile et al., 1997 ).

More generally, tobamoviruses from places as far removed as California and Crete appear to be part of one large world population with very limited variation. This remarkably constrained variation suggests that, despite varying selective pressures, the viral genome remains generally immutable as a result of long-term host–virus interactions. In other words, there appears to be a restricted window of TMV sequence variability, outside of which the host plant's ability to recognize and repulse this pathogen is greatly enhanced. In this respect, TMV appears to be very different from other viruses, such as influenza and HIV, which characteristically exhibit high rates of nucleotide change. The restricted variation characterizing TMV worldwide likely aided early virologists, who were able to duplicate results from distant laboratories with relative ease.

Invading pathogens have generally evolved to insinuate themselves into the metabolic pathways of their hosts and adapt these pathways for their own use. Thus, studies of virus–plant interactions are not only valuable for furthering understanding of the viral life cycle and the mechanisms of viral diseases, but they also shed light on such general cellular processes as the regulation of gene expression, hormonal responses, intercellular communication, and molecular transport. Historically, TMV has often served as the experimental system of choice for these lines of research. Indeed, as Harrison noted, F.C. Bawden and N.W. Pirie first suggested in 1936 that virus replication might be analogous to the synthesis of cellular components.

Several speakers at the symposium demonstrated that this analogy has continued to inform research on plant cell biology. Joseph Atabekov (Moscow State University, Russia) discussed viral functions involved in intercellular movement. Specifically, TMV spreads from cell to cell through plasmodesmata until it reaches the vascular system, which mediates long-distance transport. The spread of TMV through the vasculature may be a primarily passive process, occurring with the flow of photoassimilates. In contrast, cell-to-cell movement requires specific interactions between virus components and plasmodesmata. Such interactions are mediated in the case of TMV by the MP ( Figure 1 ), which acts to increase plasmodesmal permeability and to facilitate transport of viral genomic RNA through these enlarged channels. Surprisingly, TMV can also mediate the movement of unrelated viruses, such as potato leaf roll luteovirus (PLRV), which are normally limited to the host phloem ( Atabekov and Taliansky, 1990 ).

Research into the ability of TMV to promote virus nonspecific cell-to-cell movement has focused on the TMV MP, and this research has contributed broadly to our understanding of plant intercellular communication. Vitaly Citovsky (State University of New York, Stony Brook) described his initial discovery that the TMV MP specifically binds to single-stranded nucleic acids, and he surmised that the MP directly attaches to viral RNA in a sequence-nonspecific manner to facilitate plasmodesmal transport. Citovsky went on to report the recent identification of a 38-kD tobacco cell wall protein (p38) which specifically binds to TMV MP. Two MP domains are involved in p38 recognition, and these regions were previously shown to be required for viral movement and gating of plasmodesmata ( Gafny et al., 1992 ; Waigmann et al., 1994 ). He also reported that the effect of MP on plasmodesmal permeability is negatively regulated by phosphorylation of serine and threonine residues near its C terminus ( Citovsky et al., 1993 ). Citovsky's presentation closed with the description of a recessive single gene mutation in Arabidopsis, termed vsm1 , which blocks the systemic spread of TMV ( Lartey et al., 1998 ), and he suggested that this mutant might be useful in efforts to elucidate the general mechanisms of intercellular molecular movement in plants.

Bill Dawson (University of Florida, Lake Alfred) offered an overview of attempts by researchers during the past century to identify the causes of symptoms associated with TMV infection. Dawson recalled that Beijerinck first observed that TMV produces a disease of chloroplasts, a finding that was pursued by F.C. Bawden in the 1930s when he linked chlorosis to specific stages of TMV infection and disease ( Bawden, 1939 ). Dawson intrigued symposium participants with his ability to predict the onset of systemic vein clearing with an accuracy of about 20 min. Dawson's ‘magic box’ showed that, counter to virologists' intuition, vein clearing may not be a direct consequence of virus infection, but rather can result from the rapid physiological signaling that precedes viral invasion of the upper leaves. These results may relate to molecular genetic studies showing that the mosaic phenotype ( Atkinson and Matthews, 1970 ) is dependent on the amino acid composition of the TMV replicase proteins ( Lewandowski and Dawson, 1993 ). Dawson's presentation illustrated the perspective gained by combining the traditional physiological and pathological concepts in TMV biology with the insights realized from modern cellular and molecular approaches.

Speaking of more modern approaches, Roger Beachy (The Donald Danforth Plant Science Center, St. Louis, MO) discussed the recent contributions that TMV research has made toward elucidating the mechanism of pathogen-derived host protection, a topic that illustrates the reciprocating interests of basic science on the one hand and commercial incentives on the other. Beachy reminded the audience that resistance against TMV in CP-expressing transgenic plants seems to be a protein-mediated process in which the cytoplasmic accumulation of CP subunits somehow affects the disassembly of incoming TMV particles ( Bendahmane et al., 1997 ). Through a sophisticated combination of reverse genetics and powerful computing procedures, experiments in Beachy's laboratory are beginning to shed light on the intricate structural interactions involved in CP-mediated protection. More generally, the achievements of his laboratory since the 1980s have paved the way for numerous other strategies for engineering pathogen-derived resistance. These biotechnological innovations have in turn advanced our understanding of cosuppression and transgene silencing, events that have provided headaches (and opportunities) for many researchers working with transgenic plants.

Barbara Baker (United States Department of Agriculture–Plant Gene Expression Center, Albany, CA) offered an update on studies of the N gene, which provides gene-for-gene resistance against TMV. The significance of this discovery became apparent upon the demonstration that the transgenic introduction of the N gene into tomato plants confers the ability to activate a TMV-specific hypersensitive response ( Whitham et al., 1996 ). Although N is a single gene in tobacco, Baker reported recent results suggesting that alternative splicing of the N transcript plays a role in mediating resistance, and she outlined the ongoing evaluations of postulated molecular interactions between the product of the N gene and the viral replicase protein.

The ascendance of biotechnology in the 1980s and 1990s has reoriented TMV research toward commercial application, as Wilson pointed out. For example, the TMV Ω-leader sequence has been utilized to substantially enhance the translation of certain transgenes ( Gallie et al., 1987 ). From Wilson's subsequent summary of recent progress in the development of virus vectors, it became apparent that TMV is playing a leading role in the exploration of various strategies to use viruses as vectors for expressing foreign genes in plants. Tom Turpen (Biosource Technologies, Vacaville, CA) echoed the current interest in TMV not only as a model system, but also as a commercial vector. The high titer of TMV in infected plants and the renowned stability of the virus contribute to its attractiveness for use in large-scale production of highly valued compounds. Turpen reported that Biosource has used TMV to produce therapeutic human enzymes of high purity and specific activity, and that pilot tests are underway in new biomass processing facilities in Kentucky for the harvesting of commercially valuable proteins from field-grown tobacco plants. One could not fail to reflect upon the socio-economic and political implications of “milking tobacco leaves for human enzymes,” especially when it is happening in “tobacco country”.

These recent biotechnological advances encompass the entire history of research on TMV, from its discovery in an agricultural context to its most modern practical applications. Lute Bos (Wageningen Agricultural University, The Netherlands) argued that one century ago the convergence of agricultural concerns over “mosaic infected” tobacco and the emerging germ theory of disease gave Beijerinck's discovery of TMV its resounding scientific impact ( Bos, 1995 ). At the beginnings of virology as a discipline, TMV helped researchers define what a virus was, and throughout the twentieth century, experimentation on this model virus has led scientists to a greater understanding of both life and disease.

Current developments in TMV research are equally auspicious. The exploitation of the TMV genome, the molecular mining of natural reservoirs of genetic resistance, and the use of viruses as molecular tools, represent promising and potentially very powerful avenues of investigation. Even in this modern era of accelerating appreciation for molecular mechanisms, TMV research maintains its status as a pioneering endeavor to advance our understanding of biology.

The authors acknowledge research support through grants from the National Science Foundation (Grant. No. SBR9412291 to A.N.H.C.), the National Institutes of Health (Grant No. R01-GM50224 to V.C.), the U.S. Department of Agriculture (Grant Nos. CS-REES-NRI-CGP 94-02564 to V.C., 96-35303-3714 to K.-B.G.S., and 95-37303-2289 to H.B.S.), the U.S.–Israel Binational Research and Development Fund (BARD) (Grant No. US-2247-93 to V.C.), and the Texas Agricultural Experiment Station (Grant Nos. H-8388 to K.-B.G. S. and H-8387 to H.B.S.). Readers might be interested to know that a collection of papers based on contributions to the meeting will be published under the title “Tobacco Mosaic Virus: Pioneering Research for a Century” on March 29, 1999 (Phil. Trans. Royal Soc. Lond. Ser. B, Vol. 354). Also, an anthology of seminal TMV papers entitled “Tobacco Mosaic Virus: One Hundred Years of Contributions to Virology” edited by K.-B.G. Scholthof, J.G. Shaw, and M. Zaitlin is scheduled for publication in the spring of 1999 by the American Phytopathological Society Press (St. Paul, MN).

Abel P.P.   Nelson R.S.   De B.   Hoffmann N.   Rogers S.G.   Fraley R.T.   Beachy R.N. ( 1986 ). Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene . Science   232 , 738 – 743 .

Google Scholar

Anderer F.A.   Uhlig H.   Weber E.   Schramm G. ( 1960 ). Primary structure of the protein of tobacco mosaic virus . Nature   186 , 922 – 925 .

Atabekov J.G.   Taliansky M.E. ( 1990 ). Expression of a plant virus-coded transport function by different viral genomes . Adv. Virus Res.   38 , 201 – 248 .

Atkinson P.H.   Matthews R.E.F. ( 1970 ). On the origin of dark green tissue in tobacco leaves infected with tobacco mosaic virus . Virology   40 , 344 – 356 .

Bawden F.C.   Pirie N.W.   Bernal J.D.   Fankuchen I. ( 1936 ). Liquid crystalline substances from virus-infected plants . Nature   138 , 1051 – 1055 .

Bawden F.C. ( 1939 ). Plant Viruses and Virus Diseases . ( Leiden, The Netherlands : Chronica Botanica Company ).

Google Preview

Beachy R.N.   Zaitlin M.   Bruening G.   Israel H.W. ( 1976 ). A genetic map for the cowpea strain of TMV . Virology   73 , 498 – 507 .

Beijerinck M.W. ( 1898 ). Über ein contagium vivum fluidum als Ursache der Fleckenkrankheit der Tabaksblätter . Verh. K. Akad. Wet. Amsterdam   65 , 3 – 21 . (Translation published in English as Phytopathological Classics No. 7 [1942], American Phytopathological Society, St. Paul, MN.)

Bendahmane M.   Fitchen J.H.   Zhang G.   Beachy R.N. ( 1997 ). Studies of coat protein–mediated resistance to tobacco mosaic tobamovirus: Correlation between assembly of mutant coat proteins and resistance . J. Virol.   71 , 7942 – 7950 .

Bos L. ( 1995 ). The embryonic beginning of virology: Unbiased thinking and dogmatic stagnation . Arch. Virol.   140 , 613 – 619 .

Bloomer A.C.   Champness J.N.   Bricogne G.   Staden R.   Klug A. ( 1978 ). Protein disk of tobacco mosaic virus at 2.8 Å resolution showing the interactions within and between subunits . Nature   276 , 362 – 368 .

Butler P.J.G. ( 1984 ). The current picture of the structure and assembly of tobacco mosaic virus . J. Gen. Virol.   65 , 253 – 279 .

Caspar D.L.D. ( 1956 ). Radial density distribution in the tobacco mosaic virus particle . Nature   177 , 928 .

Caspar D.L.D. ( 1960 ). The structural stability of tobacco mosaic virus . Trans. N.Y. Acad. Sci.   22 , 519 – 521 .

Citovsky V.   Knorr D.   Schuster G.   Zambryski P. ( 1990 ). The P30 movement protein of tobacco mosaic virus is a single-stranded nucleic acid binding protein . Cell   60 , 637 – 647 .

Citovsky V.   McLean B.G.   Zupan J.R.   Zambryski P. ( 1993 ). Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall–associated protein kinase . Genes Dev.   7 , 904 – 910 .

Crick F.C.   Watson J.D. ( 1956 ). Structure of small viruses . Nature   177 , 473 – 475 .

Dawson W.O.   Beck D.L.   Knorr D.A.   Grantham G.L. ( 1986 ). cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts . Proc. Natl. Acad. Sci. USA   83 , 1832 – 1836 .

Deom C.M.   Oliver M.J.   Beachy R.N. ( 1987 ). The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement . Science   237 , 389 – 394 .

Domingo E.   Holland J.J. ( 1994 ). Mutation rates and rapid evolution of RNA viruses . In The Evolutionary Biology of Viruses , Morse S.S. , ed ( New York : Raven Press ), pp. 161 – 184 .

Eriksson-Quensel I.-B.   Svedberg T. ( 1936 ). Sedimentation and electrophoresis of the tobacco-mosaic virus protein . J. Am. Chem. Soc.   58 , 1863 – 1867 .

Fraenkel-Conrat H.   Williams R.C. ( 1955 ). Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components . Proc. Natl. Acad. Sci. USA   41 , 690 – 698 .

Fraenkel-Conrat H. ( 1956 ). The role of the nucleic acid in the reconstitution of active tobacco mosaic virus . J. Am. Chem. Soc.   78 , 882 – 883 .

Fraenkel-Conrat H.   Singer B. ( 1957 ). Virus reconstitution. II. Combination of protein and nucleic acid from different strains . Biochim. Biophys. Acta   24 , 540 – 548 .

Fraile A.   Escriu F.   Aranda M.A.   Malpica J.M.   Gibbs A.J.   García-Arenal F. ( 1997 ). A century of tobamovirus evolution in an Australian population of Nicotiana glauca . J. Virol.   71 , 8316 – 8320 .

Franklin R.E. ( 1955 ). Structure of tobacco mosaic virus . Nature   175 , 379 – 381 .

Franklin R.E. ( 1956 ). Location of the ribonucleic acid in the tobacco mosaic virus particle . Nature   177 , 928 – 930 .

Franklin R.E.   Klug A.   Holmes K.C. ( 1956 ). X-ray diffraction studies of the structure and morphology of tobacco mosaic virus . In Ciba Foundation Symposium on The Nature of Viruses , Wolstenholme G.E.W.   Millar E.C.P. , eds ( London, UK : Churchill Press ), pp. 39 – 55 .

Gafny R.   Lapidot M.   Berna A.   Holt C.A.   Deom C.M.   Beachy R.N. ( 1992 ). Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus . Virology   187 , 499 – 507 .

Gallie D.R.   Sleat D.E.   Watts J.W.   Turner P.C.   Wilson T.M.A. ( 1987 ). The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo . Nucleic Acids Res.   15 , 3257 – 3273 .

Gierer A.   Schramm G. ( 1956 ). Infectivity of ribonucleic acid from tobacco mosaic virus . Nature   177 , 702 – 703 .

Gierer A.   Mundry K.-W. ( 1958 ). Production of mutants of tobacco mosaic virus by chemical alteration of its ribonucleic acid in vitro . Nature   182 , 1457 – 1458 .

Goelet P.   Lomonossoff G.P.   Butler P.J.G.   Akam M.E.   Gait M.J.   Karn J. ( 1982 ). Nucleotide sequence of tobacco mosaic virus RNA . Proc. Natl. Acad. Sci. USA   79 , 5818 – 5822 .

Heinlein M.   Epel B.L.   Padgett H.S.   Beachy R.N. ( 1995 ). Interaction of tobamovirus movement proteins with the plant cytoskeleton . Science   270 , 1983 – 1985 .

Hunter T.R.   Hunt T.   Knowland J.   Zimmern D. ( 1976 ). Messenger RNA for the coat protein of tobacco mosaic virus . Nature   260 , 759 – 764 .

Ivanowski D. ( 1892 ). Über die Mosaikkrankheit der Tabakspflanze . Bull. Acad. Imp. Sci. St. Petersb., Nouv. Sér.   3 : 35 , 67 – 70 . (Translation published in English as Phytopathological Classics No. 7 [1942], American Phytopathological Society, St. Paul, MN.)

Jackson A.O.   Zaitlin M.   Siegel A.   Francki R.I.B. ( 1972 ). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separate leaf cells . Virology   48 , 655 – 665 .

Joshi S.   Pleij C.W.A.   Haenni A.L.   Chapeville F.   Bosch L. ( 1983 ). The nature of the tobacco mosaic virus intermediate length RNA-2 and its translation . Virology   127 , 100 – 111 .

Kausche G.A.   Pfankuch E.   Ruska H. ( 1939 ). Die Sichtbarmachung von pflanzlichem Virus im Übermikroskop . Naturwissenschaften   27 , 292 – 299 .

Kay L.E. ( 1998 ). A book of life? How the genome became an information system and DNA a language . Persp. Biol. Med.   41 , 504 – 526 .

Lartey R.T.   Ghoshroy S.   Citovsky V. ( 1998 ). Identification of an Arabidopsis thaliana mutation ( vsm1 ) that restricts systemic movement of tobamoviruses . Mol. Plant-Microbe Interact.   11 , 706 – 709 .

Lauffer M.A.   Ansevin A.T.   Cartwright T.E.   Brinton C.C. ( 1958 ). Polymerization—depolymerization of tobacco mosaic virus protein . Nature   181 , 1338 – 1339 .

Lewandowski D.J.   Dawson W.O. ( 1993 ). A single amino acid change in tobacco mosaic virus replicase prevents symptom production . Mol. Plant-Microbe Interact.   6 , 157 – 160 .

Mayer A. ( 1886 ). Über die Mosaikkrankheit des Tabaks . Landwn. Vers-Stnen.   32 , 451 – 467 . (Translation published in English as Phytopathological Classics No. 7 [1942], American Phytopathological Society, St. Paul, MN.)

McLean B.G.   Zupan J.   Zambryski P. ( 1995 ). Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells . Plant Cell   7 , 2101 – 2114 .

Meshi T.   Ishikawa M.   Motoyoshi F.   Semba K.   Okada Y. ( 1986 ). In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus . Proc. Natl. Acad. Sci. USA   83 , 5043 – 5047 .

Namba K.   Pattanayek R.   Stubbs G. ( 1989 ). Visualization of protein–nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9Å resolution by x-ray fiber diffraction . J. Mol. Biol.   208 , 307 – 325 .

Nishiguchi M.   Motoyoshi F.   Oshima N. ( 1978 ). Behaviour of a temperature sensitive strain of tobacco mosaic virus in tomato leaves and protoplasts . J. Gen. Virol.   39 , 53 – 61 .

Ohno T.   Takamatsu N.   Meshi T.   Okada Y.   Nishiguchi M.   Kiho Y. ( 1983 ). Single amino acid substitution in 30K protein of TMV defective in virus transport function . Virology   131 , 255 – 258 .

Palukaitis P.   Zaitlin M. ( 1986 ). Tobacco mosaic virus: Infectivity and replication . In Plant Viruses. Vol. 2. The Rod-Shaped Plant Viruses , Van Regenmortel M.H.V.   Fraenkel-Conrat H. , eds ( New York : Plenum Press ), pp. 105 – 131 .

Pelham H.R.B. ( 1978 ). Leaky UAG termination codon in tobacco mosaic virus RNA . Nature   272 , 469 – 471 .

Stanley W.M. ( 1935 ). Isolation of a crystalline protein possessing the properties of tobacco mosaic virus . Science   81 , 644 – 645 .

Tsugita A.   Gish D.T.   Young J.   Fraenkel-Conrat H.   Knight C.A.   Stanley W.M. ( 1960 ). The complete amino acid sequence of the protein of tobacco mosaic virus . Proc. Natl. Acad. Sci. USA   46 , 1463 – 1469 .

Waigmann E.   Lucas W.   Citovsky V.   Zambryski P. ( 1994 ). Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability . Proc. Natl. Acad. Sci. USA   91 , 1433 – 1437 .

Watanabe Y.   Emori Y.   Ooshika I.   Meshi T.   Ohno T.   Okada Y. ( 1984 ). Synthesis of TMV-specific RNAs and proteins at the early stage of infection in tobacco protoplasts: Transient expression of the 30 K protein and its RNA . Virology   133 , 18 – 24 .

Watson J.D. ( 1954 ). The structure of tobacco mosaic virus. I. x-Ray evidence of a helical arrangement of sub-units around the longitudinal axis . Biochim. Biophys. Acta   13 , 10 – 19 .

Whitham S.   McCormick S.   Baker B. ( 1996 ). The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato . Proc. Natl. Acad. Sci. USA   93 , 8776 – 8781 .

Wilson T.M.A. ( 1984 ). Cotranslational disassembly of tobacco mosaic virus in vitro . Virology   137 , 255 – 265 .

Wolf S.   Deom C.M.   Beachy R.N.   Lucas W.J. ( 1989 ). Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit . Science   246 , 377 – 379 .

Wu X.   Shaw J.G. ( 1996 ). Bidirectional uncoating of the genomic RNA of a helical virus . Proc. Natl. Acad. Sci. USA   93 , 2981 – 2984 .

Wu X.   Shaw J.G. ( 1997 ). Evidence that a viral replicase protein is involved in the disassembly of tobacco mosaic virus particles in vivo . Virology   239 , 426 – 434 .

Zaitlin M. ( 1998 ). The discovery of the causal agent of the tobacco mosaic virus disease . In Discoveries in Plant Biology , Kung S.D.   Yang S.F. , eds. ( Hong Kong : World Scientific Publishing Co. Ltd. ), pp. 105 – 110 .

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Department of History

The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930-1965

We normally think of viruses in terms of the devastating diseases they cause, from smallpox to AIDS. But in The Life of a Virus , Angela N. H. Creager introduces us to a plant virus that has taught us much of what we know about all viruses, including the lethal ones, and that also played a crucial role in the development of molecular biology. Focusing on the tobacco mosaic virus (TMV) research conducted in Nobel laureate Wendell Stanley's lab, Creager argues that TMV served as a model system for virology and molecular biology, much as the fruit fly and laboratory mouse have for genetics and cancer research. She examines how the experimental techniques and instruments Stanley and his colleagues developed for studying TMV were generalized not just to other labs working on TMV, but also to research on other diseases such as poliomyelitis and influenza and to studies of genes and cell organelles. The great success of research on TMV also helped justify increased spending on biomedical research in the postwar years (partly through the National Foundation for Infantile Paralysis's March of Dimes)—a funding priority that has continued to this day.

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Practicing virology: making and knowing a mid-twentieth century experiment with Tobacco mosaic virus

Karen-beth g. scholthof.

1 Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132 USA

Lorenzo J. Washington

2 Present Address: Plant and Microbial Biology, University of California, Berkeley, CA USA

April DeMell

3 Present Address: Plant Biology, University of California, Davis, CA USA

Maria R. Mendoza

4 Present Address: FujiFilm Diosynth Biotechnologies, College Station, TX USA

Will B. Cody

5 Present Address: Chemical Engineering, Stanford University, Stanford, CA USA

Tobacco mosaic virus (TMV) has served as a model organism for pathbreaking work in plant pathology, virology, biochemistry and applied genetics for more than a century. We were intrigued by a photograph published in Phytopathology in 1934 showing that Tabasco pepper plants responded to TMV infection with localized necrotic lesions, followed by abscission of the inoculated leaves. This dramatic outcome of a biological response to infection observed by Francis O. Holmes, a virologist at the Rockefeller Institute for Medical Research, was used to score plants for resistance to TMV infection. Our objective was to gain a better understanding of early to mid-twentieth century ideas of genetic resistance to viruses in crop plants. We investigated Holmes’ observation as a practical exercise in reworking an experiment, having been inspired by Pamela Smith’s innovative Making and Knowing Project. We had a great deal of difficulty replicating Holmes’ experiment, finding that biological materials and experimental customs change over time, in ways that ideas do not. Using complementary tools plus careful study and interpretation of the original text and figures, we were able to rework, yet only partially replicate, this experiment. Reading peer-reviewed manuscripts that cited Holmes’ 1934 report provided an additional level of insight into the interpretation and replication of this work in the decades that followed. From this, we touch on how experimental reworking can inform our strategies to address the reproducibility “crisis” in twenty-first century science.

If a photograph is worth a thousand words, then we were taken (in) by an image from a 1934 scientific manuscript in the journal Phytopathology (Fig.  1 ). The figure shows a Tabasco pepper leaf dropping from the plant following inoculation with Tobacco mosaic virus (TMV). Tabasco plants respond to TMV infection within a few days of inoculation, first with localized necrotic lesions (LNLs) on the inoculated leaf. The LNLs are mere pinpoints, oftentimes all but obscured by the damage incurred by rub-inoculation. Leaf abscission occurs a few days after LNLs are observed. This response—to sacrifice an inoculated leaf to rid itself of the virus—is a dramatic outcome. Francis O. Holmes, a virologist at the Rockefeller Institute for Medical Research, used both responses to monitor for the presence of a dominant gene for resistance to TMV infection. 1

Photograph showing the effects of TMV-infection on homozygous ( ll ) and heterozygous ( LL of Ll) plants from the genetic cross of Tabasco X bell pepper. The figure caption reads: “Two plants of Capsicum frutescens , inoculated with tobacco-mosaic virus. The first was a mottling-type plant and the second, a necrotic-type. A. 3 days after inoculation of 2 leaves each. B. 7 days after inoculation. Inoculated leaves had fallen from necrotic-type plant, freeing it from virus [arrow added for emphasis]. C. 16 days after inoculation. Mottling-type plant was stunted and mottled. Necrotic-type one was large, without symptoms, and free of virus” (Holmes, 1934 , p. 988). The notations of symptom type, mottling ( ll ) and localized necrotic lesions ( Ll and LL plants); and the days post-inoculation (dpi) with TMV were added to clarify Holmes’ experimental results. (Holmes, 1934 , Fig.  2 , p. 988, used with permission of the American Phytopathology Society.)

We were interested in replicating this experiment as an exemplar of “practicing virology” within the context of the history of science. Our work, initially inspired by Pamela Smith’s Making and Knowing Project, was fraught with challenges. A seemingly simple experiment belied the complexity and challenges of reworking an experiment from the past. We concluded that some experiments from the past cannot be replicated in full; that complementary methods are oftentimes necessary to interpret experimental results across the decades; that careful and attentive reading and interpretation of text and figures is necessary and essential to rework an experiment; and, identification and reading manuscripts that cite the original work is an extremely useful tool to interpretate historical experiments. Here we discuss our challenges and successes with reference to findings from historians of science who have reworked interesting experiments of the past. We also touch on the role of craft (making) and pitfalls associated with biological materials for historical reworking (knowing). In relation to a perceived “reproducibility crisis” in recent science, we discuss, in light of our experience, potential difficulties in reworking experiments, which includes identifying, replicating, funding and publishing the results. Finally, we hope our experiments will encourage more hands-on reworking as a key component of the historiography of the life sciences because of its informative value.

Practicing virology

Prior to the rediscovery and wide-spread acceptance of Mendelian genetics, crop improvement was based on observation. Plant pathologists and breeders would survey fields, collecting seeds of plants with desirable traits, such as improved yield, or escape from the ravages of diseases. This seed would be increased and used in subsequent seasons. Another, more focused strategy, evaluated seed from local collections or that provided by the USDA. 2 With Mendelian genetics, plant breeders in the twentieth century could deliberately introduce new, desirable traits to crop plants. Such “inheritable traits could be charted through mathematical probabilities” allowing for “efficient and predictable” outcomes including genetic resistance to plant pathogens (Campbell et al., 1999 , p. 257). Seeds were harvested from plants with the desired phenotypes, followed by pathogen challenge of a new generation of (hybrid) plants. Plants tolerant or resistant to the challenge were advanced through the trials, grown to maturity and their seed harvested. Plants from these seeds, were backcrossed to plants with commercially desirable features. A stable genetic line would be developed with nearly all the original “good” features of a parent plant with the addition of genetic resistance to a particular pathogen. This work could take years. (While todays molecular methods allow for more rapid identification of the resistance genes, the breeding process remains labor and time intensive.) Finally, the seed would be increased for commercial use. 3

Tobacco mosaic virus was one pathogen causing economic losses in tobacco, pepper and tomato fields. In the early twentieth century, understanding the “nature” of the virus was an enormously difficult task as viruses could not be cultured or observed by light microscopy. By necessity, indirect methods were developed to study viruses and their interactions with host plants (Fig.  2 ). Francis O. Holmes was a scientist who is now recognized for creating innovative and reproducible advances in virology and plant breeding in the early twentieth century, first at the Boyce Thompson Institute for Plant Research (Yonkers, NY), then the Rockefeller Institute for Medical Research (Princeton, NJ). He reported on the development of a biological assay for plant viruses that involved the visualization of TMV infection on tobacco and other plants (Holmes, 1929b ) (Fig.  2 ). Holmes observed small LNLs accumulating on TMV-inoculated Nicotiana glutinosa leaves. The virus was confined within the boundaries of the lesions on the inoculated leaf—this host response was protective, allowing the tobacco plant to complete its lifecycle without detriment. Holmes determined this response was due to N. glutinosa harboring a single dominant gene ( N ) for resistance to TMV infection (Holmes, 1929b , 1931 ; Scholthof, 2004 , 2011 , 2014 ). Then, he used Mendelian techniques to cross the N. glutinosa gene- N into N. tabacum (tobacco) as a first step to develop commercial tobacco lines with field resistance to TMV (Holmes, 1934 , 1938 ; Scholthof, 2014 , 2016 ). 4 The LNL response to TMV infection was used as a biological assay to confirm the introgression of the N -gene into tobacco plants.

Illustration of mechanical inoculation of plant viruses as shown in the 2nd edition of Plant Pathology , a textbook by George N. Agrios ( 1978 ) used by generations of plant pathologists. (Agrios, 1978 , Fig. 213, p. 568, used with permission of Elsevier.)

This process was fraught with difficulties in that it took Holmes three years to advance this project (Holmes, 1934 , 1938 ; Scholthof, 2014 , 2016 ). During this experimental interregnum Holmes pursued a similar approach with pepper ( Capsicum species), finding it more amenable to Mendelian breeding strategies. 5 He had determined that Tabasco pepper leaves inoculated with TMV developed small LNLs, then dropped from the plant a few days later (Fig.  1 ), rendering the plant virus free. Holmes attributed this effect to the presence of the Tabasco gene L , analogous to the N. glutinosa N -gene. With this knowledge, Holmes incorporated the Tabasco gene L into commercial lines of bell pepper, thus protecting the plants from systemic TMV infection. 6 Today, this same L -gene is found in TMV-resistant bell pepper cultivars. The 1934 publication is important to plant pathology because it was the first demonstration that a resistance gene from one species could be used to protect another species from the ravages of virus infection. As shown by Holmes, Tabasco pepper leaves abscised within days of TMV inoculation, a striking means to visualize a gene-in-action in the pre-molecular biology era.

The manifest issues of technique, skill, tools, and temporal distance have been addressed by Pamela Smith’s pathbreaking “Making and Knowing Project” at Columbia University. Smith has commented on “how odd it is that historians whose object of study is historical materials and techniques … have generally not considered engagement with the materials of their historical topics as an essential part of their training and research” (Smith, 2016 , p. 9). Here, acting as scientists and practitioner-historians, we investigate a historical topic and the value of tacit (or gestural) knowledge in experiment and interpretation. We concur with Smith that making and knowing is an “necessary part of our intellectual toolbox ... through hands-on work with materials and techniques,” and that the devil is in the details—some of which, as we will show, are details that we had initially not considered (Smith, 2016 , p. 9). We explore, through demonstration, the complexity of “doing biology” across the decades. We found that although ideas travel, the biological components (plants, viruses) and performance of a technique are more difficult to locate. 7

In his TEA set paper Harry Collins addressed the difficulty of replication across physical distance, even for those expert in their area of practice and craft (Collins, 1974 ). 8 Collins interviewed TEA laser scientists, finding that peer-reviewed publications and citations were used to suggest “the flow of articulated and therefore visible information,” but this did not give a full understanding of “the modes of transfer of real, useable knowledge among a set of scientists” (Collins, 1974 , pp. 170, 174). We are attempting to develop and construct parameters to transfer information—an experiment (Fig.  1 ). For Collins, the trial and error aspects of developing a new technology (tool) and “the non-systematic element” (Collins, 1974 , p. 175) were part of the process of making and knowing, something we also encountered in setting up this “simple” TMV experiment.

Here, we provide an example of reproducing knowledge at a temporal distance, using a biological experiment. We encountered many of the same problems mentioned in the TEA set paper as we worked to reproduce an experiment from a written document. Collins indicated that “written sources … as the sole source of information” are inadequate and the ability to reproduce an experiment or build a piece of equipment or “reinvent it” indicates that the (naïve) group “knows as much” as the reporter (Collins, 1974 , p. 176). Pamela Smith pulls these ideas together in a material framework (Smith, 2012 ). The “how to” comes about with deliberate reading, interpretation, testing, experimentation, and analysis of the results. In all instances repetition is key to mastering each step in the reworking—the craft of becoming a “maker”. Interpretation, analysis and extension of the findings is “knowing”. This iterative process entails significant time, material resources, hands-on experience, mistakes, troubleshooting, and critical thinking.

Several scholars have been at the forefront in engaging in the “experimental history of science” (Fors et al., 2016 , p. 89) to deepen our understanding of the insight, craft, practice, and ideas of early physical and chemical scientists (Ahnfelt & Fors, 2016 ; Ahnfelt et al., 2020 ; Albala, 2010 ; Barwich & Rodriguez, 2020 ; Bilak, 2020 ; Chang, 2011 ; Fors et al., 2016 ; Hendriksen, 2020 ; Hendriksen & Verwaal, 2020 ; Principe, 1987 ; Root-Bernstein, 1983 ; Sibum, 1995 ; Smith, 2012 ; Usselman et al., 2005 ). Their contextualization of the historiography through experimentation brings us a richer understanding of scientific processes, development, and epistemology. Yet, little reworking has occurred within the life sciences.

One example of biological reworking was a counting study by Robert Root-Bernstein. This project revealed the difficulty of reproducing a seemingly straightforward problem in biology: identification by observation of seed characteristics (the phenotype) using maize kernels (Root-Bernstein, 1983 ). As described by Root-Bernstein, the early twentieth century controversy surrounding the results of Mendel’s garden pea study (when 1936 Ronald Fisher proclaimed that the counting must be off or that some fudging occurred because it surely was not possible to have those precise predicted ratios) could be resolved by a simple experiment. Instead of using peas, Root-Bernstein selected maize, using a monohybrid cross (pure lines of purple seed X yellow seed parents); the hybrid would produce, according to Mendel, an equal ratio of purple:yellow kernels. He asked undergraduate students to count the number of purple or yellow kernels on an ear. Root-Bernstein found that it is more difficult to assess a phenotype (the physical expression of a gene) than expected, with upwards of 2% of the kernels “indeterminant” or “difficult to classify”. However, the general results were in line with what was predicted by Mendelian ratios. A more difficult task of scoring two dihybrid crosses with the “traits purple, yellow, wrinkled and smooth,” classified 6% of the kernels as “indeterminate” (Root-Bernstein, 1983 , p. 284).

Root-Bernstein’s work leads us to a similar experiment reported by Raymond Pearl in 1911 (Pearl, 1911 ). Pearl used “fifteen trained observers” who “were required to discriminate only with reference to the color [yellow or white] and the form [starchy (smooth) or sweet (wrinkled)] of each kernel” with the expected Mendelian second generation ratios of 9 yellow starchy:3 yellow sweet:3 white starchy:1 white sweet (Pearl, 1930 , p. 127). 9 All observers counted 532 kernels, yet none of the “highly trained and competent observers” were in agreement concerning the distribution of the characteristics (Pearl, 1930 , p. 129). Pearl wrote that this “seems a simple problem. One only has to count them. They [the kernels] do not run away or change” (Pearl, 1930 , p. 129). This was a reworking at the most simple state – no preparation of plants, chemicals, inoculation, or cultivation. Merely counting. Root-Bernstein found that with practice, the students became better at making choices and in which bin to place the kernels. This outcome reminds us of the comment by Barbara McClintock to Evelyn Fox Keller that there is “a feeling for the organism” or, something that develops over time, allowing the experimentalist to ‘see’ and ‘understand’ more deeply with immersion than as a novice (Keller, 1984 ). We suggest that this is the beauty of reworking experiments. Making and knowing allows us to understand more about the methods and conclusions reported on by historical actors, the constraints associated with materials available in a given time period, and the experiential skills needed to accomplish fundamental, interesting studies in the sciences. The TMV-pepper experiment seemed an ideal project to learn more about Holmes’ ideas and his standard practices.

Making: materials and methods

The impetus for this experiment was the dramatic image of pepper leaf abscission several days following the LNL response to TMV inoculation, as shown by Holmes (Holmes, 1934 ) (Fig.  1 ). Our objective was to gain a better understanding of early to mid-twentieth century ideas of genetic resistance to viruses in crop plants. 10 Identifying the working biological materials (TMV strains and pepper plants) used by Holmes for his experiments was non-trivial. With only the briefest textual description of his methods in his published scientific papers, we had to interpret the experimental design.

Plants and planting

We purchased Tabasco and Heirloom California Wonder (sweet bell pepper) seeds from W. Atlee Burpee & Co. Two TMV-susceptible tobacco lines, N. tabacum cv. Turk and N. benthamiana (commonly used for laboratory experiments), were cultivated from our laboratory seed stock. 11 All plants were grown using the conditions shown in Table ​ Table1 1 . 12

Known variables based on Holmes’ pepper experiment

a A question mark (?) indicates a best guess of the source based on manuscripts, reports and correspondence

b TMV U1 strain is available from the American Type Culture Collection ( https://www.atcc.org ), catalog number PV-135

c Tabasco (22,661) and bell pepper seeds California Wonder (60816A), Bullnose (64495A), Chinese Giant (51888A) were sourced from W. Atlee Burpee Co., 2018–2019 catalogs ( www.burpee.com )

d Also “outdoor conditions in the [Boyce Thompson] Institute gardens” (Holmes, 1932 , p. 323)

e Promix (PBPGX28) growing medium with sphagnum peat moss (75–85%), vermiculite, limestone, and wetting agent was sourced from Premier Tech Horticulture ( www.pthorticulture.com )

f General purpose 20–20-20 (N-P-K) fertilizer from J. R. Peters (G99290; www.jrpeters.com ). Plants were treated weekly with 0.25 ppm fertilizer in water

g Plant age for Holmes’ experiment was determined from the photograph shown in Fig.  1 , adapted from Holmes ( 1934 )

h Celite is powdered diatomaceous earth used as an abrasive to damage the leaf, allowing virus entry into the cells

Commercially produced seed introduces additional genetic variables, although the plants may appear to be identical (phenotype). For example, a genetic analysis of ten lines of California Wonder showed the plants could be grouped into 5 classes, based on genetic polymorphisms identified by PCR amplification with a series of primer sets to randomly sample the genome (Votava & Bosland, 2002 ). The authors cautioned that California Wonder “exists in name only” and its utility as a standard control should be determined based on the type of experiments performed (Votava & Bosland, 2002 , p. 1101). 13 Similarly, for Tabasco ( C. frutescens ) it is not possible to definitively state that the plant is identical to Holmes’ Tabasco; almost certainly it is not the same. 14 However, from observations made by Walter H. Greenleaf, a plant breeder and pathologist at Auburn University (Alabama), we know that “the L -gene in peppers provides an effective form of resistance” to “all tested strains of TMV from tobacco and tomato” (W. H. Greenleaf, 1986a , 1986b , p. 98), giving us a degree of confidence that a commercially available of Tabasco would be suitable for these experiments.

Rub inoculation

In the late 1920s, in a series of experiments, Holmes developed the rapid and efficient inoculation technique, now a standard practice, known as mechanical (or rub) inoculation (Holmes, 1928 , 1929a , 1929b , 1931 ) (Fig.  2 ). For rub-inoculation, one or more TMV-infected symptomatic N. tabacum leaves were pulverized with the addition of water or phosphate buffer (1:10 w/v), using a mortar and pestle. The negative control experiment, or mock-inoculation utilizes healthy leaves. Two or three lower leaves of a plant are rubbed gently with the sap extract following dusting with an abrasive powder (carborundum or Celite) to slightly injury the leaf, allowing virus ingress (Kalmus & Kassanis, 1945 ). Immediately after inoculation the leaves were rinsed with water. The plants were observed every day and symptoms were recorded, with particular attention to local lesions and systemic infections. 15

Tobacco mosaic virus (TMV)

The TMV common strain (U1) was maintained on N. tabacum cv. Turk and N. benthamiana . This strain induces necrotic local lesions on N. glutinosa and Tabasco pepper. Pepper leaves were rub-inoculated following Holmes’ method (Fig.  2 ). Unfortunately, due to rub inoculation damage on our plants, it was difficult to count lesions and to determine the level of infection. To rework this experiment we used a more tractable tool, an infectious TMV cDNA construct. This is a routine plant molecular virology practice to determine if pepper plants were susceptible to TMV infection. 16 Specifically, our complementary experiment utilized a molecular construct of TMV with the addition of the green fluorescent protein ( gfp ) gene (TMV-GFP) (Fig.  3 A). TMV-GFP infected tobacco leaves were harvested and used as inoculum for the pepper experiments (Fig.  2 ). 17 TMV-GFP was used to i) monitor virus infection (count local lesions) by fluorescence under ultraviolet light and ii) determine the sites of virus replication versus inoculation damage. TMV-GFP provided consistent and genetically homogeneous inoculum to investigate TMV infection, development of LNLs, and leaf abscission.

Exploring Holmes’ results with Tobacco mosaic virus (TMV) and pepper plants with the techniques of molecular biology. a The molecular genetic map of TMV with the addition of a reporter gene encoding the green fluorescent protein (GFP). The rectangles indicate protein-encoding genes of TMV: replicase, movement protein (MP), and capsid protein (CP). The bent arrows indicate the subgenomic RNA promoters. The asterisk indicates that a specialized strategy of readthrough translation to express two replicase proteins from the genomic RNA. b and c Representative Tabasco pepper ( Capsicum frutescens ) and Nicotiana tabacum cv. Turk (tobacco) leaves at 2, 3 and 4 days post-inoculation with TMV-GFP. The leaves were photographed under white light and ultraviolet (UV) light. In Fig. 3B, rub-inoculation damage of the inoculated leaves presents as brown discoloration under white light and greyish-white discoloration under UV light. The same leaves were used for white and UV light exposure. The localized green fluorescent spots on Tabasco and tobacco leaves reflect single infection events following inoculation with TMV-GPF, equivalent to the localized necrotic lesions reported by Holmes. On tobacco, the pinpoint florescence spots at 2 dpi become much larger by 4 dpi, indicating TMV resistance gene N is not present. In time these green fluorescent spots coalesce and progress to systemic infection (not shown). D. California Wonder bell pepper ( C. annuum ) plants showing systemic infection at 24 days post-inoculation with TMV-GFP

Other historians of science who had pursued their own reworking of experiments, reported using modern tools as they developed their craft (Ahnfelt & Fors, 2016 ; Ahnfelt et al., 2020 ; Albala, 2010 ; Barwich & Rodriguez, 2020 ; Bilak, 2020 ; Chang, 2011 ; Fors et al., 2016 ; Hendriksen & Verwaal, 2020 ; Principe, 1987 ; Root-Bernstein, 1983 ; Sibum, 1995 ; Usselman et al., 2005 ). For example, Hasok Chang uses modern instruments to understand historical experiments. For his “complementary” experiments on the boiling point of water, he explained “when practitioners of historical replication say they try to get ‘as close to the original as possible’, that is usually with a clear awareness of some inherent limits to faithfulness. It is not always possible to match exactly the past instruments and operations described in historical papers” (Chang, 2011 , p. 320). Chang also notes the historic manuscript may exclude some methodology because it was “well-understood by readers in the original context” (Chang, 2011 , p. 320). For these reasons, we introduced complementary molecular virology tools as “ opportunities for better historical understanding” (Chang, 2011 , p. 321).

Knowing: results

We rub-inoculated two or three leaves of small Tabasco and bell pepper plants with sap of TMV or mock-inoculated plants for the control experiment. Our expectation was that visible chlorotic lesions would develop on California Wonder bell pepper leaves within 7 to 10 days, followed by mottling of the upper, non-inoculated leaves. For Tabasco pepper, we expected to observe LNLs within a few days, followed by leaf abscission. Instead, in our hands, abscission was observed for mock- and TMV-inoculated bell pepper and Tabasco plants.

We were especially confounded by the abscission response we observed on mock-inoculated leaves. Holmes consistently emphasized that rapid leaf drop was a marker for the Tabasco L -gene. For example, TMV-infected L -gene segregating bell pepper lines, such as California Wonder, “show necrotic primary lesions only, and their inoculated leaves were soon lost by abscission” (Holmes, 1937 , p. 641). 18 However, Holmes also reported that older plants as well as plants maintained under different environmental conditions may defoliate independent of L -gene-associated abscission (Holmes, 1932 , p. 352). Altogether, we decided to focus our attention on (1) environmental conditions; (2) inoculation techniques, (3) confirming the mock-inoculated plants were not contaminated with TMV; and (4) the possibility of genetic variability of California Wonder, such as the inclusion of the L -gene or minor resistance genes or inadvertent contamination of seed lots.

As we know from Holmes, California Wonder is susceptible to TMV infection (Table ​ (Table2 2 ). 19 Yet genetic variability within California Wonder occurs, as reported by Eric Votova and Paul Bosland, pepper breeders at New Mexico State University. This variability is a result of inadvertent mixing of seed lots by producers, intentional selection by plant breeders over time, or genetic drift (Votava & Bosland, 2002 ). Of course, it is impossible to rework the experiment with the exact same seeds Holmes used, which may have affected our interpretation of his findings (Table ​ (Table2). 2 ). However, we did determine that California Wonder was susceptible to TMV (Fig.  3 D). We then narrowed our considerations to environmental conditions and plant age.

Summary of effects of tobacco mosaic virus infection on Capsicum (pepper) plants (Holmes, 1937 )

a Genetic resistance to TMV infection was ranked by Holmes using the notation LL  >  l i l i  >  ll (Holmes, 1937 )

b Commercial bell pepper, such as California Wonder, was described as large fruited, blocky shaped, non-pungent. Plants with the l i l i background had “imperfect localization” of TMV, showing “delayed necrosis with leaf abscission” (Holmes, 1937 , p. 642). The l i l i allele was associated with some commercial bell pepper varieties, including Ruby King. In the field, TMV-infected pepper plants with the ll allele were mottled and stunted, with significant reduction in yield and quality

c Days post-inoculation with TMV (dpi)

Early on we observed leaf drop in almost all peppers—this was particularly evident when there were changes in the environment, including decreased temperatures in the growth chamber, or lab, due to power failures or maintenance issues, or biological contamination of the growth chambers with insects and fungi (a complication of working in shared spaces in a plant pathology department). Our first estimation of plant age was based on plant height and the approximate leaf size (Fig.  1 ; 4-inch diameter clay pots). We returned to Fig.  1 and determined that Holmes had used more mature plants, based on a count of the visible internodes. From this, we decided it would be worthwhile to test older plants for the abscission response.

Doing it again: laboratory practice and practicing

Plant virus inoculation and the molecular biology technique of the plasmid prep (isolating plasmid DNA from bacteria, generally E. coli ), are both considered straightforward “ubiquitous practice” (Jordan & Lynch, 1992 , p. 78). These methods of practice are so basic that they are used in undergraduate laboratory exercises (Dijkstra & De Jager, 1998 ; Ford & Evans, 2003 ). As elaborated by Kathleen Jordan and Michael Lynch, seemingly rote processes are predicated on more than the ability to read a protocol. Oftentimes there are “persistent problems associated with establishing the coherence and efficacy of the practice, determining whether one practitioner’s method for doing it is the same as another’s, accounting for discrepant results, and explaining how the technique works” (Jordan & Lynch, 1992 , p. 77). Importantly, this is in spite of the protocol being “relatively standardized, reproducible, coherent, and subject to rational reconstruction” (Jordan & Lynch, 1992 , p. 77). Yet protocols are neither rational or standardized without technique—typically acquired through apprenticeship. Here we are evaluating two aspects of a “mundane practice” (Jordan & Lynch, 1992 , p. 78): i) are Holmes’ observations reproducible in our hands? And ii) what sort of expertise matters to recapitulate previously published data?

In Jordan and Lynch’s study, they interrogated practitioners to learn about differences in a common practice, asking about variation “between their own and others’ methods” as well as “local circumstances of the lab and idiosyncrasies of its members” (Jordan & Lynch, 1992 , p. 78). Like the plasmid prep, virus inoculation is a key, mundane practice that must be learned (Figs.  1 , ​ ,2 2 and ​ and3). 3 ). Pamela Smith and Tonny Beentjes discuss this “makers’ knowledge” within the context of reconstructing life-casting techniques in the sixteenth-century. They emphasized that the “knowledge possessed by handworkers, also known as ‘makers’ knowledge’” is key to understanding the materials, techniques and “how and why nature was investigated” (Smith & Beentjes, 2010 , p. 130).

The “simplicity” of TMV inoculation of tobacco is made evident by its common use as an experiential tool for in plant pathology laboratory courses (Dijkstra & De Jager, 1998 ; Ford & Evans, 2003 ). Yet, rub inoculation is a particular practice subject to many errors, including damaging plants by rubbing leaves with too much enthusiasm (Fig.  3 A). The experimental outcome “can depend on the particular ingredients used, as well as an endless array of other circumstantial features” (Jordan & Lynch, 1992 , p. 81), even for a virus inoculation method standardized in the mid-1930s. 20

We systemically compared our materials and methods to those reported by Holmes (Table ​ (Table1) 1 ) and identified many variables, some of which may have affected the outcome of our reworking experiments. For example, in our hands pepper was exquisitely sensitive to environmental conditions, especially changes in ambient temperature. When we returned to the text, making a more careful study of his publications we found that Holmes had reported that TMV-susceptible Capsicum (and several other plant species) exposed to cooler growing temperatures may experience premature leaf abscission (Holmes, 1932 , p. 337). Another identified variable was plant age. When carefully inspecting Fig.  1 , we noticed that Holmes’ plants had several internodes, indicating more mature plants. In our subsequent reworking experiments, we used older plants. But our plant growth conditions resulted in tall plants with with elongated internodes, a result of low light intensity (Fig.  4 ). The variables that had foiled our initial efforts encompassed the key determinants of infection: the host, the virus, and the environment. Parsing the most important variables towards becoming proficient with Holmes’ methods, we realized the experimental protocol had features that were strikingly similar to those mentioned by Jordan and Lynch: “Although the plasmid prep is far from controversial and is commonly referenced as a well-established and indispensable technique, how exactly it is done is not effectively communicated, either by print, word of mouth or demonstration. Instead, it is mastered largely through repeated (and often solitary) practice” (Jordan & Lynch, 1992 , p. 84).

Recapitulation of the Tabasco pepper experiments described by Holmes. Panels a , b , and c . Wildtype TMV inoculated to Tabasco, as shown in Fig.  1 , and photographed at 3, 7 and 15 days postinoculation (dpi). On Tabasco leaves the necrotic pinpoint local lesions are difficult to observe, especially when the leaves are damaged during inoculation. b and c The TMV-inoculated leaf abscission noted at 7- and 15-dpi on two plants; mock inoculated leaves at 7 dpi have not abscised. An “X” on the leaf indicates that the leaf was inoculated (TMV or mock). In B, the center figure is a close up of the dropped leaf shown in the leftmost photograph. These results can be compared to those Holmes ( 1934 ), shown in Fig.  1

Initially this seemed a straightforward project to gain some understanding about how Holmes performed his experiments and if we could achieve similar results. What we know now is that these seemingly trivial experiments were fraught with technical difficulty and a great deal of complexity, even though we were merely pulverizing a TMV-infected leaf, rubbing it on a healthy pepper leaf and observing the outcomes of infection (mottling, leaf drop, etc.). The choices we and Holmes made were not trivial or insignificant. As discussed by Jordan and Lynch in their analysis of the standardized protocol for plasmid preps: “For practitioners at the bench, these distinctions [choices] do not easy resolve such issues as what to include or exclude from a procedure, what to do now, and what to do next” (Jordan & Lynch, 1992 , p. 100). We were handicapped by the lack of detailed protocols and an ever increasing number of parameters to attend to. Upon reflection, the lack of detailed materials and methods is not a Holmes-specific issue, nor is it an issue limited to historical biological reworking.

Reading between the lines: reproducibility

Our difficulty in reworking a decades-old experiment gets at broader questions. Is it possible to reproduce an experiment? Is an experiment valid if it never is reproduced? Perhaps the reproducibility is what makes co-discoveries from different labs so exciting—the realization that a phenomenon is “real” and others notice it as well. This suggests that there is merit to multiple groups tackling similar questions. These issues are of considerable interest as the science community has faced concerns about the reproducibility of peer-reviewed data and, in extensively corrected or retracted papers, if the remaining data has value in a given manuscript. 21

The early outcomes of our experimental reworking were both frustrating and dissatisfying. We had success in observing localized necrotic lesions and abscission, but wondered if abscission was the gold-standard bioassay to score peppers for TMV resistance. Did contemporaries of Holmes confirm his findings or report useful modifications that we were unaware of when we initiated our experimental reworking? Is this method used today to evaluate TMV resistance in Capsicum ?

This sort of closer reading and (re-) interpretation is integral to science practice. For example, Staffan Müller-Wille and Giuditta Parolini inspected copies of Mendel’s pea breeding manuscript, finding that readers “actively engaged with the text” by “rehearsing calculations and by employing Mendel’s notation system” (Müller-Wille & Parolini, 2020 , p. 157). The annotations and underlining revealed what the reader “deemed most important” (Müller-Wille & Parolini, 2020 , p. 153). Today, genetics students learn to use Punnett’s square for visualizing the outcome of genetic crosses of dominant and recessive genes. This “interplay between text and image” (Müller-Wille & Parolini, 2020 , p. 163) and annotation, for us, was an important part of a process that revealed Holmes’ ideas, to identify experimental materials and methods, and to design complementary experiments. Later, we repeated this process as a tool to troubleshoot possible errors in reworking the pepper experiment. As Müller-Wille and Parolini write, this “active engagement” is a fundamental aspect of research “alongside the observations conducted at the lab bench and the experimental garden” to interpret and gain “practical knowledge” (Müller-Wille & Parolini, 2020 , pp. 164, 165). Similarly, Pamela Smith showed us the importance engaging with material objects, text and drawings, to recreate skills and knowledge of the past (Smith, 2012 ). Nils-Otto Ahnfelt and Hjalmar Fors also reported that they “should have returned to the sources and read the original recipes more carefully”; consulted other sources to inform a practice or provide an “indirect pointer” for troubleshooting and problem-solving; and, performed “complementary” experiments with modern instrumentation to replicate the historical work (Ahnfelt & Fors, 2016 , p. 177).

To evaluate the reproducibility of Holmes’ experiment in others’ hands, across a gap of nine decades, we identified 17 manuscripts, from 1936 to 2021, that reported on TMV inoculation of L -gene pepper plants and also cited Holmes’, 1934 or 1937 Phytopathology papers (Table ​ (Table3). 3 ). Of these papers, all 17 reported LNLs following inoculation of L -gene pepper plants with TMV; 12 also reported leaf abscission. Two authors reported they obtained L -gene bell pepper seed from Holmes (Greenleaf, 1953 ; Murakishi, 1960 ). Harry Murakishi, at Michigan State University, used Holmes’ “LL-resistant garden pepper”, reporting LNLs appeared following TMV inoculation, but he did not indicate leaf abscission (Murakishi, 1960 , p. 464). Walter H. Greenleaf, a pepper breeder at Auburn University, regularly exchanged seed and viruses with Holmes, and on several occasions reported that Holmes’ LL -bell pepper responded to TMV inoculation with LNLs and abscission (Greenleaf, 1953 , 1986a , 1986b ). Greenleaf also used Holmes’ L -gene bell pepper line to develop a TMV-resistant pimiento pepper (Greenleaf et al., 1969 ).

Citation analysis of selected manuscripts published by F. O. Holmes ( 1929a , 1929b , 1934 , 1937 , 1938 ) a

a Analysis performed using Web of Science ( https://clarivate.com ) with search parameters “Holmes FO (Author) and 1929–1938 (Year Published)”. The number of peer-reviewed articles citing the original publications are shown with citation data. Four journal articles were used for the citation analyses (Holmes, 1929a , 1929b , 1934 , 1937 , 1938 )

b Year of publication of the four papers used by Holmes used for citation analysis (Holmes, 1929a , 1929b , 1934 , 1937 , 1938 )

c Tobacco indicates Nicotiana species, pepper indicates Capsicum species; LNLs refers to localized necrotic lesions on an inoculated leaf following TMV infection; abscise indicates the inoculated leaf drops from the plant following TMV infection; and, L -gene and N -gene refer to dominant resistance gene in Tabasco pepper and Nicotiana glutinosa , respectively, introgressed into crop plant varieties

d Total citations of the paper in the decade following publication. The parentheses indicate the number of self-citations (self-cites) by Holmes in subsequent publications for that decade (a subset of total citations)

e Total citations from year of publication through October, 2021

f Both papers citing Holmes were on the general topic plant (pepper) breeding, not the experimental use of TMV

H. H. McKinney, a USDA plant virologist, found that TMV-inoculated Capsicum frutescens ( L -gene) plants maintained at 23ºC developed “local necrotic lesions on mature and nearly mature leaves … and these leaves eventually absciss” (McKinney, 1937 , p. 55). Similarly, Glenn S. Pound and G. P. Singh at the University of Wisconsin, noted “necrotic lesions on inoculated leaves. At all temperatures, inoculated leaves abscised and the plants remained free of systemic infection” (Pound & Singh, 1960 , p. 805). In 1968, Mo-Yeong Lee and Paul G. Smith at the University of California-Davis, scored pepper lines for TMV-resistance by the LNLs response “just before leaf abscission” (Lee & Smith, 1968 , p. 1445). But twelve papers, most of which did not cite Holmes' TMV-pepper work, did not mention abscised leaves in response to TMV infection, suggesting that it was not a consistently reliable assay (as we found) or it added no additional value to the standard scoring for LNLs.

Donna Bilak and her colleagues in the Making and Knowing Project, remind us that “recipe literature is a challenging genre to read, not only because of its frequent technical obscurity and abridged prose, but often even more so because of its simple style and apparent straightforwardness” (Bilak et al., 2016 , p. 41). The same can be said the materials and methods sections of peer-reviewed scientific manuscripts and protocol manuals. 22 Ken Albala, a culinary historian and practitioner, reminds us of the importance of Renaissance cooks who “recorded their extensive experience” even if the methods are unfamiliar to us; in short, “we must trust what is on the page” (Albala, 2010 , p. 87). Similarly, the experimental work of Lawrence Principe reveals that reconstruction of the alchemy is possible because the work is “grounded in chemical reality, even though a simple reading of the text by a person well-versed in chemistry might well suggest the contrary” (Principe, 1987 , p. 27). Yet, peer-reviewed manuscripts often have insufficient details to reproduce experiment. And becoming adept with new techniques and tools, such as construction of a TEA laser (Collins, 1974 ), may require communication with the innovators, spending many frustrating weeks to years to become expert in the method, or waiting for a commercial company to develop a kit, machine, or service to standardize the technique.

In our pursuit of a decades old experiment using established, standard methodology we found nearly every element (soil, watering regimen, plants, lighting, inoculum, and pest control measures) affected our ability to make and learn from Holmes. This has ramifications for scientists and historians who decide to replicate key experiments in their field. As we show here, and as discussed by Jordan and Lynch for plasmid preps, success with a particular protocol belies the depth of required experience and expertise by the users. Our difficulty in recapitulating Holmes’ work was difficult and interesting, and we learned a few things:

1. We became better readers and observers. As noted by Pamela Smith and others, we too stumbled on the processual research, providing us with the opportunity (and necessity) of studying processes rather than discrete events, to carefully read the materials and methods, and then assemble the needed reagents and tools. This extends into the need for carefully reading the protocols following failed attempts. In the early stages, the process of re-investigation seemed straightforward. Yet it quickly became evident that we were missing or unable to identify the materials used by Holmes. We interpreted what we expected to observe; that is, we anticipated ‘seeing’ the exact outcomes shown in Fig.  1 , but we had not realized that a particular combination of plant age, lighting and temperature would determine the outcome. We did not review the literature citing Holmes, as we were intent on reworking his experiment. However, this literature revealed that LNLs were sufficient to identify TMV-resistance plants. Leaf abscission offered no added value when studying L -gene pepper plants or developing new commercial varieties.

2. Side projects are projects. The TMV-pepper experiment was piggy-backed onto ‘normal’ experiments in our molecular virology laboratory. We do not often perform experiments unrelated to our primary research interests. “What is typical, rather, are extended series of experiments which communicate among each other with different intensity and constitute an experimental texture,” as noted by Hans-Jorg Rheinberger (Rheinberger, 2001 , p. 53). The TMV experiment dislocated us from our normal science practice, re-enforcing that that tacit knowledge and its implicit peculiarities are relevant to the success of the practitioner (Keller, 1984 ). We greatly underestimated the time and effort required to rework this experiment, because we were confident that the experiments were simple, and could be managed as a ‘side project’. Instead, we found that it takes time, money, and intensive focus to rework an experiment—there are no shortcuts.

Repeating this ‘simple’ experiment cast doubt on our expertise, leading us to revert to our familiarity with recombinant DNA tools to visualize the results. In Jordan and Lynch’s study of the plasmid prep, interviews with practitioners made evident that there is both a “black box” aspect and a “reflective” aspect to this work, and an individual in a laboratory (and an individual laboratory), may have strong feelings “over just what sorts of variations are tolerable, trivial, or significant” (Jordan & Lynch, 1992 , p. 105). It is inevitable that we work with available materials and these may change over time (plant lines, virus strains), specific conditions (laboratory infrastructure) and expertise (Holmes’ experiences versus our experiences). From the outlines and framework presented by Holmes, we have re-realized the complexity of our ‘everyday’ work in the laboratory. The devil is in the details.

3. Reproducibility is experimentation. We were humbled by the complexity of repeating an experiment from 1934. What we found, reiterating the analysis by other re-workers in the making and knowing community, is that written materials and methods are important, but are not technical guides or how-to manuals to replicate an experiment. The repetition, frustration, and mastery of techniques are part and parcel of doing science. Of equal importance are the ineffable influences of mentors and peers, institutions, classroom knowledge and laboratory training and a dash of serendipity that affect the successes, failures, interpretation and presentation of data.

The difficulty of repeating this work speaks to a larger issue in the biological science—reproducibility. Reproducibility in science has relevance to scientists, historians, philosophers, publishers and funding agencies. Journals provide guidelines to authors, emphasizing that the materials and methods should be sufficiently detailed for the work to repeated or replicated. 23 Yet, from our ‘simple’ experimental reworking, the written manuscript was not sufficient—crucial information was found in a photograph (Fig.  1 ). Perhaps this not surprising, because images (drawings, photographs, video, and models) “are especially effective in organizing technical knowledge into an abbreviated form” (Smith, 2012 , p. 24).

Pamela Smith and Hasok Chang have shown us through their historiographic reconstructions that the text and even drawings are not enough; making is process of “observation and imitation” of experts, oftentimes requiring complementary experiments (Chang, 2011 ; Smith, 2012 , p. 10). The repetitive nature of doing science, familiar to any laboratory researcher, is normal science (Jordan & Lynch, 1992 ). This “repeated trial and error was ‘skill’” acquired by attention and focus, such as hands-on laboratory experiences (Smith, 2012 , p. 26). Historical reconstructions, whether of artisan crafts, counting seeds, measuring the boiling point of water, or TMV-inoculation, can be used to address what is perceived to be a reproducibility “crisis” in science. In every instance, historiographic reconstruction has shown the impracticability of exactness in reproducing written work. For us, as scientists and practitioner-historians, we have been excited by how closely our reworking reflects ‘normal science’ by using journal articles, protocol manuals, and in-house experience to plan, perform and evaluate an experiment. Along the way modifications and changes occur, sometimes becoming normalized practice in a laboratory. Scientific manuscripts outline how work was performed, they are narratives of new findings, on the path towards new, even significant, advances in a field of study. If the work is subjected to replication then, by the very nature of scientific practice, somewhat different outcomes may be reported. Hasok Chang, using the term “extension” pushes this point, as we interpret it: Does the historian become a scientist, or considered to be practicing science when the reworking or complementary experiments lead to “something new (though old) about nature” or “genuine original contributions to scientific knowledge”? (Chang, 2011 , p. 324). And, vice versa: are scientists who reproduce recently published findings from another laboratory acting as historians of science?

Another aspect of reproducibility is choice. Replicative experiments do not have the scientific prestige of original work, despite the fact that the financial outlay (salaries, reagents, equipment, and publication costs) is equivalent to discovery-based research. Which experiments will be tested for reproducibility? For a specific example, which of 200,000 COVID-19 manuscripts published the past 18 months should be replicated? 24 As shown by Johan Chu and James Evans, most “scholarly attention” is focused on highly cited papers from well-known labs, making it difficult for “less-established papers—even those with novel, useful, and potentially transformative ideas” to gain attention (Chu & Evans, 2021 ).

Of papers subjected to replication, how one approaches an experiment is predicated on many parameters, including biased approaches and interpretations. 25 The influence(r)s guiding the reworking of a particular experiment; interpreting and troubleshooting results; and which findings should be emphasized will differ—even for scientists working together. For example, our reworking of Holmes’, 1934 experiment could be judged unsuccessful: our plants did not look exactly the same, we did not have identical results, and we relied on complementary methods. But, upon reflection, our self-analyses was too harsh. In fact, we learned about the complexity of reworking by localizing temporal and material constraints, identifying decades of changes to “normal” science (training, tools and regents) and making use of advances in virology to understand Holmes’ findings. Importantly, peer-reviewed manuscripts that cited Holmes work, showed us that the LNLs assay, not abscission, remained the standard by which plants were (and are) scored for resistance to TMV.

Local knowledge and placelessness

In their exploration of allosteric regulation, Angela Creager and Jean-Paul Gaudillière focused on the role of local knowledge and the co-evolution of meaning and experiments within individual locations (Creager & Gaudillière, 1996 , p. 90). We interpreted Holmes’ meaning and intent as we reworked his experiment. Robert Kohler has stated that laboratories “are simplified and standardized, stripped of all context and environmental variations; they are places apart from the world—placeless places. It is this odd spatial quality that gives knowledge produced in labs its credibility. The simplicity and sameness of labs helps ensure that experiments turn out the same wherever they are done, which is one of the main reasons why we trust experiment more than other ways of knowing” (Kohler, 2002 , p. 191). Yet we did not experience this, and such difficulties in repeating what may be considered normalized science call into question Robert Kohler’s idea of a laboratory as a “placeless place”. As with Creager and Gaudillière’s historiography of allosteric regulation experiments in Berkeley and Paris, we (and Holmes) “worked with different systems, local habits, and distinctive strategies for making decisions” (Creager & Gaudillière, 1996 , p. 3). Although we “envisioned” we were “working on the same problems and being part of the same group,” in our case studying TMV, the “decisions made and observations found in each setting affected choices and possibilities” (Creager & Gaudillière, 1996 , p. 3). Thus, we had to temper our expectations as we worked to replicate Holmes’ experiments.

In our instance, we were separated not by an ocean, but by time. To perform our experiments we made assumptions about Holmes’ experiments across a gap of decades, yet we “envisioned” ourselves as working on the same problem. Our experiments were informed by Holmes, reconstructing as best we could, his materials and methods. We obtained similar, but not identical results. We learned that location, practice, and a “feeling” for the tools/objects/agents matter greatly when re-working and re-assessing any experiment of the past (Chang, 2011 ; Creager & Gaudillière, 1996 ; Keller, 1984 ; Kohler, 2002 ).

Again, from Smith, we were reminded that “experiential” or makers’ knowledge is gained by the experimental habit of “doing things over and over” and she (and we) “marveled at the length of time it took to acquire experiential knowledge”(Smith, 2012 , pp. 22–23). We found that reworking Holmes’ experiment differed little from initiating a new project including the attendant pitfalls, problem-solving, and interpretation of the results—we had to become wholly immersed in the process of practice. That Holmes intuited the presence of a host resistance gene to TMV infection from an observation of localized necrotic lesion and leaf abscission, shows us a scientist who mastered the craft of working with his research tools, to make foundational advances in virology.

Acknowledgments

This work was funded in part by a National Science Foundation SES grant (No. 1456878) awarded to K.-B.G.S. W.B.C. was funded by an USDA-NIFA Predoctoral Award. We thank Angela Creager and Herman Scholthof for helpful, critical comments as we prepared this manuscript, as well the suggestions from the two anonymous referees.

Author’s contributions

KBGS developed the project and wrote the manuscript; WBC, LJW, AD, and MRM performed the experiments; all authors contributed to the analysis of results, developing the figures, and manuscript edits.

1 For recent scholarship on the history of tobacco mosaic virus and F. O. Holmes’ contributions to advances in plant virology, see Creager ( 2002 ), Creager & Morgan ( 2008 ), Scholthof ( 2004 , 2014 , 2016 ), Scholthof & Peterson ( 2006 ), Scholthof et al. (1999).

2 The importance of the efforts of the US government and scientists to collect and distribute seed to plant breeders and farmers in the United States has been addressed by Campbell et al., (1999), Curry ( 2016 , 2019 ), Fitzgerald ( 1990 ), Fullilove ( 2017 ), Kingsbury ( 2011 ), Kloppenburg Jr. (2005), Palladino ( 1991 ).

3 For all the successes attributed to plant breeding and genetic resistance to pathogens, a history has yet to be written of the formative years of scientific breeding for plant disease resistance. Textbooks on plant genetics and plant breeding and journals such as Phytopathology , Crop Science , Agronomy and Genetics are helpful in understanding the state-of-the art in the early twentieth century to the present. “Plant Pathology: Problems and Progress, 1908–1958” provides an overview of advances in plant pathology (Holton et al., 1959 ); in this volume, Holmes wrote a commentary on plant virology (Holmes, 1959 ).

4 On the science and technology of plant breeding and plant genetics in the United States in the early to mid-twentieth century, see Curry ( 2016 ), Fitzgerald ( 1990 ), Fullilove ( 2017 ), Kingsbury ( 2011 ).

5 We can speculate that this almost certainly is due to ploidy; that is, the genus Capsicum is diploid allowing for straightforward interpretation of species crosses by Mendelian genetics. In contrast, many of the species in Nicotiana are polyploid which confounds interpretation of genetic outcomes and introgression of genes of interest. We have addressed the difficulties Holmes had towards introgression of the resistance gene N to commercial tobacco from N. glutinosa (Scholthof, 2016 ).

6 By 1959, the USDA Farmers’ Bulletin referred growers to Rutgers World Beater No. 13, Yolo Wonder, Keystone Resistant Giant, and Liberty pepper lines, reporting “considerable resistance” to TMV (Boswell et al., 1959 , p. 27).

7 In “Doing Biology,” Joel Hagen, Douglas Allchin and Fred Singer used case studies or “historical episodes” of work by individual scientists “that exemplify important characteristics of scientific practice” to “more fully reveal how biology is done” within the context of “science-in-the-making” (Hagen et al., 1997, pp. vi, vii). This book is not a hands-on guide, instead it shows the complexity of science, decision making, and interpreting outcomes with the broad area of the history and sociology of science. Furthermore, common practice(s) also change. What were once standard techniques for plant pathologists are no longer learned in graduate (practicum) courses or the research laboratory. Instead, “hands on” experiences increasingly are replaced with lectures and journal clubs.

8 “TEA” refers to the Transversely Excited Atmospheric Pressure CO 2 laser, or TEA laser. In Collins’ paper the TEA set refers to the interactions between laboratories in North America and the U.K. who are have developed or are keen to develop the apparatus. Here, Collins discusses networks and networking between labs and how ideas and knowledge travel (Collins, 1974 ).

9 These recruits “included two plant pathologists, two professors of agronomy, one professor of philosophy (originally trained as a biologist), four biologists, one [human] computer, one practical corn breeder, and one professor and three assistants in plant physiology.” Two of these men by “birth, early life and education” belonged to “the ‘corn belt’ section of the country, and are thoroughly and intimately familiar with maize” and “had experience in corn judging” (Pearl, 1930 , pp. 126, 127). We relay this to emphasize the difficulty of reworking experiments; learning the craft behind the experimental technique is key to making and knowing (Smith, 2012 ).

10 It is important to note that this work on (scientific) material culture is constrained by university rules, and federal and state laws. Laboratory work with viruses and plant materials requires a permit and all lab members be trained in the use of biological materials, chemicals, and (recombinant) pathogens. In this instance, biological materials were used with the approval of the Texas A&M University Institutional Biosafety Committee (Permits IBC2016-130 and IBC2019-139).

11 As reported by Holmes ( 1934 ), TMV was maintained on tobacco ( N. tabacum ), as inoculum for the Capsicum experiments.

12 The seeds were sown in well-moistened Sungro brand potting mix and transferred to a growth chamber set at ~ 25 °C, 16 h light, 8 h dark ~ 22 °C, 60% humidity, 100–120 μmol/m 2 /s light intensity, watered 3 times/week and fertilized with 20–20-20 (N-P-K) once a week (20 ml/L). Following inoculation, the plants were placed in the laboratory on a plant growth rack at ~ 23 °C 16 h light, 8 h night, and watered 3 times/week without the addition of fertilizer.

13 Of importance to our study, Eric Votava and Paul Bosland reported “‘California Wonder’ should not be included as a standard control in other Capsicum research. … The concept of a standard control entails that the ‘control’ can be used in separate labs and in separate experiments and act as a consistent and repeatable benchmark. The dependability of a sample to act as a standard control is cast into doubt if it is shown to contain a high degree of variability or if even a high degree of variability is suspected” (Votava & Bosland, 2002 , p. 1102). In 1937, Holmes reported that not all plants of a seed lot responded similarity to infection, suggesting the possibility of a “contaminant of the seed lot” due to inadvertent mixing of seeds (Holmes, 1937 , p. 641). Rachel Ankeny and Sabina Leonelli provide an HPS-centered discourse on standard or wildtype organisms (Ankeny & Leonelli, 2021 ).

14 For example, in the USDA National Germplasm Collection, ~ 15 lines of Tabasco are identified within the Capsicum frutescens accessions ( https://npgsweb.ars-grin.gov/gringlobal/search ). Commercial seed companies then advance lines with specific traits (yield, maturity, fruit color, etc.).

15 The mock-inoculated leaves (control) and TMV-inoculated leaves of Tabasco and bell pepper were photographed several timepoints (days post-inoculation; dpi). Each independent experiment used three susceptible and three resistant plants and the experiments were repeated several times.

16 The infectious cDNA used for these experiments is based on the U1 (common or type) strain (Siegel & Wildman, 1954 ). This strain, as reported by Milton Zaitlin and H. W. Israel, from “personal recollections of C. A. Knight, W. C. Price and F. O. Holmes suggest that the original isolate used by W. M. Stanley came from J. Johnson of the Univ. of Wisconsin via L. O. Kunkel. The U1 strain (Wittmann & Wittmann-Liebold, 1963 ) and the German strain ‘vulgare’ (Wittmann-Liebold & Wittmann, 1967 ) also came from Johnson” (Zaitlin & Israel, 1975 ).

17 Specifically, we used a binary plasmid containing the coding sequence of TMV with a gfp insert (pJL24) (Lindbo, 2007 ). This plasmid was propagated in Agrobacterium tumefaciens strain GV3101, then infiltrated into N. benthamiana or N. tabacum cv. Turk leaves to initiate the transcription of the recombinant viral genome, TMV-GFP.

18 The California Wonder variety, lacking resistance to TMV, was released in 1937, having first been selected by a California grower in 1928 (Boswell, 1937 ; Votava & Bosland, 2002 ). Paul W. Bosland, the New Mexico State University pepper breeder, has documented an exhaustive list of garden catalog descriptions of pepper varieties and the year of commercial release. Bell pepper varieties were bred for TMV resistance by genetic introgression of the Tabasco L -gene. Two examples were California Wonder 300 (XP 300) a “thick walled, blocky California Wonder type” released by Asgrow Seed in 1966; and, California Wonder 300 TMR, released in 1999 by Carolina Seeds with “glossy green, thick walled fruit, which turn green to red at maturity, smooth blocky fruit, mostly 4-lobed, averaging 110 × 100 mm fruit size, 72 days to harvest” (Bosland, 2019 ). https://cucurbitbreeding.wordpress.ncsu.edu/2016/06/03/pepper-a-l/ .

19 The 2019 Burpee’s catalog description of Heirloom California Wonder did not indicate TMV resistance.

20 The rapid acceptance of Holmes’ local lesion method and the use of N. glutinosa for this bioassay by plant virologists was discussed previously (Scholthof, 2011 , 2014 ).

21 This “reproducibility crisis” is discussed by Marcia McNutt, president of the National Academies of Science and former editor-in-chief of Science (McNutt 2014 ); Jeremy Berg, also a former editor-in-chief of Science (Berg 2019 ); and, a Nature collection on “challenges in irreproducible research” at https://www.nature.com/collections/prbfkwmwvz .

22 Angela Creager provides an interesting discussion of the use of laboratory manuals by practitioners (Creager, 2020 ). This and other articles in the BJHS Themes issue “Learning by the Book: Manuals and Handbooks in the History of Science” edited by Elaine Leong, Angela Creager, and Mathias Grote is a particularly helpful volume to grasp the importance of text and its interpretation to perform and interpret science. Peer-reviewed manuscripts should, but mostly do not, include sufficiently detailed materials and methods to allow the work to be reproduced. How-to videos offer an alternative or supplemental format to communicate the use of materials and methods; an example is JoVE , a peer-reviewed journal of visual experimentation (jove.com).

23 Similarly, the National Institutes of Health (NIH) has policy and compliance guidelines for “Rigor and Reproducibility” for all grant proposals ( https://grants.nih.gov/policy/reproducibility/index.htm ). The guidelines, fully implemented in 2020, assert that the “application of rigor ensures robust and unbiased experimental design, methodology, analysis, interpretation, and reporting of results. When a result can reproduced by multiple scientists, it validates the original results and readiness to progress to the next phase of research” ( https://www.nih.gov/research-training/rigor-reproducibility ). In an extension of this, authors of a recent feature article in eLife , a highly ranked journal in the life sciences, suggested the establishment of communities of “'rigor champions' who are committed to promoting rigor and transparency in research” (Koroshetz et al., 2020 ).

24 Searching “COVID” at PubMed ( https://pubmed.ncbi.nlm.nih.gov/?term=covid ) yielded 194,281 publications on 15 November 2021. The Web of Science Core Collection ( https://clarivate.com ) identified 193,280 publications of which ca. 7,764 (4%) are highly cited (accessed 15 November 2021). “Retraction Watch” has recorded 197 retracted or problematic papers ( https://retractionwatch.com/retracted-coronavirus-covid-19-papers/ ; accessed 15 November 2021).

25 A recent report by the National Academies of Science, Engineering and Medicine (2019) addressed these topics in Reproducibility and Replicability in Science , with a particular focus on replication in chapter 7.

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• Agrios, G. N. (1978). Plant pathology . Elsevier Academic Press. Doi: 10.1016/B978-0-12-044560-8.50018-1
• Ahnfelt N-O, Fors H. Making Early Modern medicine: Reproducing Swedish bitters. Ambix. 2016; 63 :162–183. doi: 10.1080/00026980.2016.1212886. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ahnfelt N-O, Fors H, Wendin K. Historical continuity or different sensory worlds? What we can learn about the sensory characteristics of early modern pharmaceuticals by taking them to a trained sensory panel. Berichte Zur Wissenschaftsgeschichte. 2020; 43 :412–429. doi: 10.1002/bewi.202000009. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Albala K. Cooking as research methodology: Experiments in Renaissance cuisine. In: Fitzpatrick J, editor. Renaissance food from Rabelais to Shakespeare: Culinary readings and culinary histories. Ashgate; 2010. pp. 73–88. [ Google Scholar ]
• Ankeny R, Leonelli S. Model organisms. Cambridge University Press; 2021. [ Google Scholar ]
• Barwich A-S, Rodriguez M. Fashion fades, Chanel No. 5 remains: Epistemology between style and technology. Berichte Zur Wissenschaftsgeschichte. 2020; 43 :367–384. doi: 10.1002/bewi.202000006. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Berg J. Replication challenges. Science. 2019; 365 :957. doi: 10.1126/science.aaz2701. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bilak, D. (2020). Out of the Ivy and into the Arctic: Imitation coral reconstruction in cross-cultural contexts [10.1002/bewi.202000010]. Berichte zur Wissenschaftsgeschichte , 43 , 341–366. 10.1002/bewi.202000010 [ PubMed ]
• Bilak D, Boulboullé J, Klein J, Smith PH. The making and knowing project: Reflections, methods, and new directions. West 86th: A Journal of Decorative Arts Design History, and Material Culture. 2016; 23 :35–55. doi: 10.1086/688199. [ CrossRef ] [ Google Scholar ]
• Bosland, P. W. (2019). Vegetable cultivar descriptions for North America – Pepper (A-L) . https://cucurbitbreeding.wordpress.ncsu.edu/2016/06/03/pepper-a-l/
• Boswell, V. R. (1937). Yearbook of agriculture: Improvement and genetics of tomatoes, peppers, and eggplant . U.S. GPO
• Boswell, V. R., Doolittle, S. P., Pultz, L. M., Taylor, A. L., & Campbell, R. E. (1959). Pepper production, disease and insect control . US GPO http://hdl.handle.net/2027/chi.59177489
• Campbell, C. L., Peterson, P. D., & Griffith, C. S. (1999). The formative years of plant pathology in the United States . APS Press.
• Chang H. How historical experiments can improve scientific knowledge and science education: The cases of boiling water and electrochemistry. Science and Education. 2011; 20 :317–341. doi: 10.1007/s11191-010-9301-8. [ CrossRef ] [ Google Scholar ]
• Chu JSG, Evans JA. Slowed canonical progress in large fields of science. Proceedings of the National Academy of Sciences. 2021; 118 :e2021636118. doi: 10.1073/pnas.2021636118. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Collins HM. The TEA set: Tacit knowledge and scientific networks. Science Studies. 1974; 4 :165–185. doi: 10.1177/030631277400400203. [ CrossRef ] [ Google Scholar ]
• Creager ANH. The life of a virus: Tobacco mosaic virus as an experimental model, 1930–1965. University of Chicago Press; 2002. [ Google Scholar ]
• Creager ANH. Recipes for recombining DNA: A history of Molecular Cloning: A laboratory manual . BJHS Themes. 2020; 5 :225–243. doi: 10.1017/bjt.2020.5. [ CrossRef ] [ Google Scholar ]
• Creager ANH, Gaudillière J-P. Meanings in search of experiments and vice-versa: The invention of allosteric regulation in Paris and Berkeley, 1959–1968. Historical Studies in the Physical and Biological Sciences. 1996; 27 :1–89. doi: 10.2307/27757769. [ CrossRef ] [ Google Scholar ]
• Creager ANH, Morgan GJ. After the double helix: Rosalind Franklin's research on Tobacco mosaic virus. Isis. 2008; 99 :239–272. doi: 10.1086/588626. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Curry, H. A. (2016). Evolution made to order: Plant breeding and technological innovation in twentieth-century America . University of Chicago Press.
• Curry HA. Why save a seed? Isis. 2019; 110 :337–340. doi: 10.1086/703337. [ CrossRef ] [ Google Scholar ]
• Dijkstra, J., & De Jager, C. P. (1998). Protocol 1: Mechanical inoculation of plants. In Practical plant virology: Protocols and exercises (pp. 5–13). Springer-Verlag. https://link.springer.com/content/pdf/10.1007%2F978-3-642-72030-7.pdf
• Fitzgerald DK. The business of breeding hybrid corn in Illinois, 1890–1940. Cornell University Press; 1990. [ Google Scholar ]
• Ford R, Evans T. Tobacco mosaic virus. Plant Health Instructor. 2003 doi: 10.1094/PHI-K-2003-0528-01. [ CrossRef ] [ Google Scholar ]
• Fors H, Principe LM, Sibum HO. From the library to the laboratory and back again: Experiment as a tool for historians of science. Ambix. 2016; 63 :85–97. doi: 10.1080/00026980.2016.1213009. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fullilove, C. (2017). The profit of the Earth: The global seeds of American agriculture . University of Chicago Press.
• Greenleaf WH. Effects of tobacco-etch virus on peppers ( Capsicum sp.) Phytopathology. 1953; 43 :564–570. [ Google Scholar ]
• Greenleaf WH. Pepper breeding. In: Bassett MJ, editor. Breeding vegetable crops. AVI Pub. Co; 1986. pp. 67–134. [ Google Scholar ]
• Greenleaf WH. The tabasco story. HortScience. 1986; 10 :98. [ Google Scholar ]
• Greenleaf WH, Hollingsworth MH, Harris H, Rymal KS. Bighart: Improved variety of pimiento pepper. Auburn University Agricultural Experiment Station, Leaflet No. 1969; 78 :1–8. [ Google Scholar ]
• Hagen, J. B., Allchin, D., & Singer, F. (1997). Doing biology . Benjamin Cummings. doingbiology.net
• Hendriksen MMA. Rethinking performative methods in the history of science. Berichte Zur Wissenschaftsgeschichte. 2020; 43 :313–322. doi: 10.1002/bewi.202000017. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hendriksen MMA, Verwaal RE. Boerhaave's furnace. Exploring early modern chemistry through working models. Berichte Zur Wissenschaftsgeschichte. 2020; 43 :385–411. doi: 10.1002/bewi.202000005. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Holmes FO. Accuracy in quantitative work with tobacco mosaic virus. Botanical Gazette. 1928; 86 :66–81. doi: 10.1086/333873. [ CrossRef ] [ Google Scholar ]
• Holmes FO. Inoculating methods in tobacco mosaic studies. Botanical Gazette. 1929; 87 :56–63. doi: 10.1086/333924. [ CrossRef ] [ Google Scholar ]
• Holmes, F. O. (1929b). Local lesions in tobacco mosaic. Botanical Gazette , 87 , 39–55. 10.1086/333923
• Holmes FO. Local lesions of mosaic Nicotiana Tabacum L. Contributions of the Boyce Thompson Institute. 1931; 3 :163–172. [ Google Scholar ]
• Holmes FO. Symptoms of tobacco mosaic disease. Contributions of the Boyce Thompson Institute. 1932; 4 :323–357. [ Google Scholar ]
• Holmes FO. Inheritance of ability to localize tobacco-mosaic virus. Phytopathology. 1934; 24 :984–1002. [ Google Scholar ]
• Holmes FO. Inheritance of resistance to tobacco-mosaic disease in the pepper. Phytopathology. 1937; 27 :637–642. [ Google Scholar ]
• Holmes FO. Inheritance of resistance to tobacco-mosaic disease in tobacco. Phytopathology. 1938; 28 :553–561. [ Google Scholar ]
• Holmes FO. Discussion of Chapters XLV and XLVI. In: Holton CS, Fischer GW, Fulton RW, Hart H, McCallan SEA, editors. Plant pathology: Problems and progress 1908–1958. University of Wisconsin Press; 1959. pp. 521–523. [ Google Scholar ]
• Holton, C. S., Fischer, G. W., Fulton, R. W., Hart, H., & McCallan, S. E. A. (Eds.). (1959). Plant pathology: Problems and progress, 1908–1958 . Univerisity of Wisconsin Press.
• Jordan K, Lynch M. The sociology of a genetic engineering technique: Ritual and rationality in the performance of the "plasmid prep". In: Clarke AE, Fujimura JH, editors. The right tools for the job: At work in twentieth-century life sciences. Princeton University Press; 1992. pp. 77–114. [ Google Scholar ]
• Kalmus H, Kassanis B. The use of abrasives in the transmission of plant viruses. Annals of Applied Biology. 1945; 32 :230–234. doi: 10.1016/S0065-3527(08)60527-8. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Keller, E. F. (1984). A feeling for the organism: The life and work of Barbara McClintock . Henry Holt.
• Kingsbury, N. (2011). Hybrid: The history and science of plant breeding . University of Chicago Press.
• Kloppenburg Jr., J. R. (2005). First the seed: The political economy of plant biotechnology (2nd ed.). University of Wisconsin Press.
• Kohler RE. Place and practice in field biology. History of Science. 2002; 40 :189–210. doi: 10.1177/007327530204000204. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Koroshetz, W. J., Behrman, S., Brame, C. J., Branchaw, J. L., Brown, E. N., Clark, E. A., Dockterman, D., Elm, J. J., Gay, P. L., Green, K. M., Hsi, S., Kaplitt, M. G., Kolber, B. J., Kolodkin, A. L., Lipscombe, D., MacLeod, M. R., McKinney, C. C., Munafò, M. R., Oakley, B., . . . Silberberg, S. D. (2020). Framework for advancing rigorous research. eLife , 9 , e55915. Doi: 10.7554/eLife.55915 [ PMC free article ] [ PubMed ]
• Lee M-Y, Smith PG. Identification of the L gene for tobacco mosaic resistance in three pepper species. Phytopathology. 1968; 68 :1445. [ Google Scholar ]
• Lindbo, J. A. (2007). High-efficiency protein expression in plants from agroinfection-compatible tobacco mosaic virus expression vectors. BMC Biotechnology , 7 , 52. [ PMC free article ] [ PubMed ]
• McKinney HH. Virus mutation and the gene concept. Journal of Heredity. 1937; 28 :51–57. doi: 10.1093/oxfordjournals.jhered.a104309. [ CrossRef ] [ Google Scholar ]
• McNutt M. Reproducibility. Science. 2014; 343 (6168):229. doi: 10.1126/science.1250475. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Müller-Wille S, Parolini G. Punnett squares and hybrid crosses: How Mendelians learned their trade by the book. BJHS Themes. 2020; 5 :149–165. doi: 10.1017/bjt.2020.12. [ CrossRef ] [ Google Scholar ]
• Murakishi HH. A necrotic pod streak of pepper caused by tobacco mosaic virus. Phytopathology. 1960; 50 :464–466. [ Google Scholar ]
• National Academies of Sciences, Engineeering and Medicine. (2019). Reproducibility and replicability in science . The National Academies Press. 10.17226/25303 [ PubMed ]
• Palladino P. The professionalism of plant breeding in the United States: The technology of hybridisation and its consequences. Minerva. 1991; 29 :507–515. doi: 10.1007/BF01113493. [ CrossRef ] [ Google Scholar ]
• Pearl R. The personal equation in breeding experiments involving certain character of maize. Biological Bulletin. 1911; 21 :339–366. doi: 10.2307/1536152. [ CrossRef ] [ Google Scholar ]
• Pearl, R. (1930). Introduction to medical biometry and statistics (2nd ed.). W. B. Saunders. https://catalog.hathitrust.org/Record/001581750/Home
• Pound GS, Singh GP. The effect of air temperature on multiplication of tobacco mosaic virus in susceptible and resistant pepper. Phytopathology. 1960; 50 :803–807. [ Google Scholar ]
• Principe, L. (1987). “Chemical translation” and the role of impurities in alchemy: Examples from Basil Valentine's Triumph-Wagen . Ambix , 34 , 21–30. [ PubMed ]
• Rheinberger, H.-J. (2001). History of science and the practices of experiment. History and Philosophy of the Life Sciences , 23 , 51–63. [ PubMed ]
• Root-Bernstein RS. Mendel and methodology. History of Science. 1983; 21 :275–295. doi: 10.1177/007327538302100303. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Scholthof, K.-B. G. (2004). Tobacco mosaic virus: A model system for plant biology. Annual Review of Phytopathology , 42 , 13–34. Doi: 10.1146/annurev.phyto.42.040803.140322 [ PubMed ]
• Scholthof K-BG. TMV in 1930: Francis O. Holmes and the local lesion assay. Microbe. 2011; 6 :221–225. [ Google Scholar ]
• Scholthof K-BG. Making a virus visible: Francis O. Holmes and a biological assay for Tobacco mosaic virus . Journal of the History of Biology. 2014; 47 :107–145. doi: 10.1007/s10739-013-9353-0. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Scholthof K-BG. Spicing up the N gene: F. O. Holmes and tobacco mosaic virus resistance in Capsicum and Nicotiana plants. Phytopathology. 2016; 107 :148–157. doi: 10.1094/PHYTO-07-16-0264-RVW. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Scholthof, K.-B. G., & Peterson, P. D. (2006). The role of Helen Purdy Beale in the early development of plant serology and virology. Advances in Applied Microbiology , 59 , 221–241. Doi: 10.1016/S0065-2164(06)59008-2 [ PubMed ]
• Scholthof, K.-B. G., Shaw, J. G., & Zaitlin, M. (Eds.). (1999). Tobacco mosaic virus: One hundred years of contributions to virology . APS Press.
• Sibum HO. Reworking the mechanical value of heat: Instruments of precision and gestures of accuracy in early Victorian England. Studies in History and Philosophy of Science Part A. 1995; 26 :73–106. doi: 10.1016/0039-3681(94)00036-9. [ CrossRef ] [ Google Scholar ]
• Siegel A, Wildman SG. Some natural relationships among strains of tobacco mosaic virus. Phytopathology. 1954; 44 :277–282. [ Google Scholar ]
• Smith PH. In the workshop of history: Making, writing, and meaning. West 86th: A Journal of Decorative Arts . Design History, and Material Culture. 2012; 19 :4–31. doi: 10.1086/665680. [ CrossRef ] [ Google Scholar ]
• Smith, P. H. (2016). Experiments in the early modern European investigation of nature. Viewpoint: The Magazine of the British Society for the History of Science , 110 , 8–9. http://www.bshs.org.uk/publications/viewpoint
• Smith PH, Beentjes T. Nature and art, making and knowing: Reconstructing sixteenth-century life-casting techniques. Renaissance Quarterly. 2010; 63 :128–179. doi: 10.1086/652535. [ CrossRef ] [ Google Scholar ]
• Usselman M, Rocke A, Reinhart C, Foulser K. Restaging Liebig: A study in the replication of experiments. Annals of Science. 2005; 62 :1–55. doi: 10.1080/00033790410001711922. [ CrossRef ] [ Google Scholar ]
• Votava, E. J., & Bosland, P. W. (2002). A cultivar by any other name: Genetic variability in heirloom bell pepper 'California Wonder'. HortScience 37 , 1100–1102. 10.21273/HORTSCI.37.7.1100
• Wittmann HG, Wittmann-Liebold B. Tobacco mosaic virus mutants and the genetic coding problem. Cold Spring Harbor Symposia on Quantitative Biology. 1963; 28 :589–595. doi: 10.1101/SQB.1963.028.01.079. [ CrossRef ] [ Google Scholar ]
• Wittmann-Liebold B, Wittmann HG. Coat proteins of strains of two RNA viruses: Comparison of their amino acid sequences. Molecular and General Genetics. 1967; 100 :358–363. doi: 10.1007/BF00334062. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zaitlin, M., & Israel, H. W. (1975). Tobacco mosaic virus (type strain). Description of Plant Viruses, Number 157 . https://www.dpvweb.net/dpv/showdpv/?dpvno=151

• A-Z Publications

Annual Review of Phytopathology

Volume 42, 2004, review article, tobacco mosaic virus: a model system for plant biology.

• Karen-Beth G. Scholthof 1
• View Affiliations Hide Affiliations Affiliations: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843-2132; email: [email protected]
• Vol. 42:13-34 (Volume publication date September 2004) https://doi.org/10.1146/annurev.phyto.42.040803.140322
• First published as a Review in Advance on March 05, 2004
• © Annual Reviews

Tobacco mosaic virus (TMV) has had an illustrious history for more than 100 years, dating to Beijerinck's description of the mosaic disease of tobacco as a contagium vivum fluidum and the modern usage of the word “virus.” Since then, TMV has been acknowledged as a preferred didactic model and a symbolic model to illuminate the essential features that define a virus. TMV additionally emerged as a prototypic model to investigate the biology of host plants, namely tobacco. TMV also exemplifies how a model system furthers novel, and often unexpected, developments in biology and virology. Today, TMV is used as a tool to study host-pathogen interactions and cellular trafficking, and as a technology to express valuable pharmaceutical proteins in tobacco. The history of TMV illustrates how pragmatic strategies to control an economically important disease of tobacco have had unexpected and transforming effects across platforms that impinge on plant health and public health.

Tobacco mosaic virus: An RNA virus that causes mosaic disease in tobacco and similar effects in other plants, much used as an experimental subject; abbrev. TMV . ( 8 )

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Fraenkel Conrat Experiment

Fraenkel Conrat experiment was based on the evidence that RNA also carry genetic information. This was the very first experiment that was introduced to prove that RNA is also a genetic material with the help of TMV. Tobacco Mosaic Virus is composed of 6% of RNA (not DNA) and protein.

Frankel-Conrat and co-workers determined that the reconstitution of infective virus particles may occur after the association of protein subunits and RNA. Before this experiment, there were many experimental proofs that proved DNA being a genetic material. So, it was difficult for Fraenkel to prove RNA also carries a hereditary material.

Content: Fraenkel Conrat Experiment

Experimental organism.

In the year 1957, Fraenkel Conrat and Singer reconstituted viruses after mixing mutant strain’s protein with the other strain’s RNA. As a result, the new virus particles produced by the infected host plant that has the protein type produced by the RNA parent.

Fraenkel Conrat was famous for his viral research, where he studied that in some viruses (like TMV or HRV), RNA is the controlling factor for viral reproduction or reconstitution.

Fraenkel-Conrat (1957) conducted experiments on TMV to demonstrate that a few viruses contain RNA. TMV or Tobacco Mosaic Virus is a small plant virus that causes infection in solanaceous plants that appear as in the mosaic pattern.

Tobacco mosaic virus comprises a single coiled RNA encapsulated in a cylindrical protein coat. Different strains of TMV are characterized by differences in the chemical composition of their protein coats.

Step-1 : Fraenkel Conrat first developed techniques to isolate proteins and RNA of TMV by using the appropriate chemical treatments. After isolation, it was observed that the protein alone did not cause infection in the tobacco leaves. In contrast, the separated RNA molecule was sufficient enough to cause mosaic in the tobacco leaves.

Step-2 : Then Fraenkel Conrat reversed the process by mixing the protein and RNA under appropriate conditions. After mixing the protein subunits or capsomers of TMV with the RNA molecule, he observed the reconstitution or formation of complete infective TMV particles.

Step-3 : In the third experiment, Fraenkel-Conrat and Singer took two different strains of TMV (type-A and type-B). Then they separated the RNAs from the protein coats. After that, both Fraenkel-Conrat and Singer mixed proteins of one strain with the RNA of the second strain to reconstitute hybrid viruses.

After rubbing the hybrid or reconstituted viruses onto live tobacco leaves, the phenotypically and genotypically identical progeny viruses were produced similar to the parental type from which the RNA had been isolated.

Therefore, by all these experiments, Fraenkel Conrat concluded that both DNA and RNA carries genetic information. By his experiments, it was proved that the genetic information of TMV is stored in the RNA and not in the protein.

However, DNA perhaps always function as genetic material, but RNA, in most cases, is non-genetic. Only in specific systems, where the DNA is absent, RNA function as hereditary material.

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Molecule of the Month

Molecule of the Month: Tobacco Mosaic Virus

A cylindrical arrangement of proteins protects a long strand of RNA in TMV

A Helical Virus

Tmv infection.

TMV Assembly

Exploring the structure, tobacco mosaic virus (pdb entry 2tmv).

PDB entries 2tmv and 2om3 include both the protein and the RNA in an infectious TMV particle. The illustration shown here includes only 17 subunits, along with RNA that is bound to them. Notice how the RNA is bound in a groove in the protein near the center of the ring. Three nucleotides bind to each protein subunit. The image on the right shows the clusters of acidic amino acids (bright red) that are important for disassembly of the virus inside infected cells. You can click on the illustration for an interactive Jmol version. For a closer look at these acidic amino acids, take a look at the page at Proteopedia .

Topics for Further Discussion

• Most of the viruses in the PDB are composed of a spherical shell of capsid proteins. Can you find other examples of cylindrical viruses like TMV?
• Purified TMV particles are all a very uniform 300 nanometers in length. What aspects of the structure would limit the length of assembling TMV particles?

Related PDB-101 Resources

• Browse Viruses
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• H. Wang, A. Planchart and G. Stubbs (1998) Caspar carboxylates: the structural basis of tobamovirus disassembly. Biophysical Journal 74, 633-638.
• D. J. Lewandowski and W. O. Dawson (1998) Tobamoviruses. In Encyclopedia of Virology, second edition, volume 3, edited by A. Granoff and R. G. Webster, Academic Press, pages 1780-1783.
• G. Stubbs (1999) Tobacco mosaic virus particle structure and the initiation of disassembly. Philosophical Transactions of the Royal Society of London B 354, 551-557
• B. D. Harrison and T. M. A. Wilson (1999) Milestones in the research on tobacco mosaic virus. Philosophical Transactions of the Royal Society of London B 354, 521-529.
• A. Klug (1999) The tobacco mosaic virus particle: structure and assembly. Philosophical Transactions of the Royal Society of London B 354, 531-535.

January 2009, David Goodsell

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• Published: 14 April 1956

Infectivity of Ribonucleic Acid from Tobacco Mosaic Virus

• A. GIERER 1 &
• G. SCHRAMM 1  

Nature volume  177 ,  pages 702–703 ( 1956 ) Cite this article

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IN their experiments with bacteriophages, Hershey and Chase 1 have shown that only the nucleic acid component plays a part in the intracellular multiplication. There are also indications that in simple viruses containing ribonucleic acid the nucleic acid plays a dominant part in the infection. Thus, experiments with tobacco mosaic virus have shown that the protein can be changed chemically without affecting the activity and the genetic properties 2 ; recently, it was even found 3 that part of the protein can be removed from tobacco mosaic virus without destroying the activity.

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Hershey, A. D., and Chase, M., J. Gen. Physiol. , 36 , 39 (1952).

Article   CAS   Google Scholar  

Schramm, G., and Müller, H., Hoppe Seylers Z. physiol. Chem. , 266 , 43 (1940); 274 , 267 (1942). Miller, G. L., and Stanley, W. M., J. Biol. Chem. , 141 , 905 (1941); 146 , 331 (1942). Harris, J. I., and Knight, C. A., J. Biol. Chem. , 214 , 215 (1955).

Schramm, G., Schumacher, G., and Zillig, W., Nature , 175 , 549 (1955).

Article   ADS   CAS   Google Scholar  

Fraenkel-Conrat, H., and Williams, R. C., Proc. U.S. Nat. Acad. Sci. , 41 , 690 (1955).

Lippincott, J. A., and Commoner, B., Biochim. Biophys. Acta , 19 , 198 (1956).

Hart, R. G., Nature , 177 , 130 (1956).

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Immunity and protective efficacy of a plant-based tobacco mosaic virus-like nanoparticle vaccine against influenza a virus in mice.

1. Introduction

2. materials and methods, 2.1. ethics statement, 2.2. influenza viruses, 2.3. centralized gene construction and phylogenetic analysis, 2.4. expression of plant-made ha, 2.5. vaccines and immunizations, 2.6. immune correlate studies, 2.7. hemagglutination inhibition (hi) assay, 2.8. microneutralization assay (mna), 2.10. elispot assay, 2.11. influenza virus challenges in mice, 2.12. statistical analysis, 3.1. sequence analysis of ha proteins, 3.2. expression and purification of ha antigen, 3.3. igg response following immunization, 3.4. igm response following immunization, 3.5. iga response following immunization, 3.6. cross-reactive t-cell responses, 3.7. protection against historical influenza challenge, 4. discussion, 5. conclusions, supplementary materials, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

• McLean, H.Q.; Petrie, J.G.; Hanson, K.E.; Meece, J.K.; Rolfes, M.A.; Sylvester, G.C.; Neumann, G.; Kawaoka, Y.; Belongia, E.A. Interim Estimates of 2022–23 Seasonal Influenza Vaccine Effectiveness—Wisconsin, October 2022–February 2023. MMWR Morb. Mortal. Wkly. Rep. 2023 , 72 , 201–205. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Thompson, W.W.; Weintraub, E.; Dhankhar, P.; Cheng, P.Y.; Brammer, L.; Meltzer, M.I.; Bresee, J.S.; Shay, D.K. Estimates of US influenza-associated deaths made using four different methods. Influenza Other Respir. Viruses 2009 , 3 , 37–49. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lafond, K.E.; Nair, H.; Rasooly, M.H.; Valente, F.; Booy, R.; Rahman, M.; Kitsutani, P.; Yu, H.; Guzman, G.; Coulibaly, D.; et al. Global Role and Burden of Influenza in Pediatric Respiratory Hospitalizations, 1982–2012: A Systematic Analysis. PLoS Med. 2016 , 13 , e1001977. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Iuliano, A.D.; Roguski, K.M.; Chang, H.H.; Muscatello, D.J.; Palekar, R.; Tempia, S.; Cohen, C.; Gran, J.M.; Schanzer, D.; Cowling, B.J.; et al. Estimates of global seasonal influenza-associated respiratory mortality: A modelling study. Lancet 2018 , 391 , 1285–1300. [ Google Scholar ] [ CrossRef ]
• Van Kerkhove, M.D.; Hirve, S.; Koukounari, A.; Mounts, A.W.; H1N1pdm Serology Working Group. Estimating age-specific cumulative incidence for the 2009 influenza pandemic: A meta-analysis of A(H1N1)pdm09 serological studies from 19 countries. Influenza Other Respir. Viruses 2013 , 7 , 872–886. [ Google Scholar ] [ CrossRef ]
• Allen, J.D.; Ross, T.M. H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation. Hum. Vaccin. Immunother. 2018 , 14 , 1840–1847. [ Google Scholar ] [ CrossRef ]
• Jester, B.J.; Uyeki, T.M.; Jernigan, D.B. Fifty Years of Influenza A(H3N2) Following the Pandemic of 1968. Am. J. Public Health 2020 , 110 , 669–676. [ Google Scholar ] [ CrossRef ]
• Boyoglu-Barnum, S.; Ellis, D.; Gillespie, R.A.; Hutchinson, G.B.; Park, Y.J.; Moin, S.M.; Acton, O.J.; Ravichandran, R.; Murphy, M.; Pettie, D.; et al. Quadrivalent influenza nanoparticle vaccines induce broad protection. Nature 2021 , 592 , 623–628. [ Google Scholar ] [ CrossRef ]
• Dbaibo, G.; Amanullah, A.; Claeys, C.; Izu, A.; Jain, V.K.; Kosalaraksa, P.; Rivera, L.; Soni, J.; Yanni, E.; Zaman, K.; et al. Quadrivalent Influenza Vaccine Prevents Illness and Reduces Healthcare Utilization Across Diverse Geographic Regions During Five Influenza Seasons: A Randomized Clinical Trial. Pediatr. Infect. Dis. J. 2020 , 39 , e1–e10. [ Google Scholar ] [ CrossRef ]
• Lewnard, J.A.; Cobey, S. Immune History and Influenza Vaccine Effectiveness. Vaccines 2018 , 6 , 28. [ Google Scholar ] [ CrossRef ]
• Webby, R.J.; Weaver, E.A. Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses. PLoS ONE 2015 , 10 , e0140702. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lingel, A.; Bullard, B.L.; Weaver, E.A. Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine. Sci. Rep. 2017 , 7 , 14912. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Weaver, E.A.; Barry, M.A. Low seroprevalent species D adenovirus vectors as influenza vaccines. PLoS ONE 2013 , 8 , e73313. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Weaver, E.A.; Rubrum, A.M.; Webby, R.J.; Barry, M.A. Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin. PLoS ONE 2011 , 6 , e18314. [ Google Scholar ] [ CrossRef ]
• Karron, R.A.; Key, N.S.; Sharfstein, J.M. Assessing a Rare and Serious Adverse Event Following Administration of the Ad26.COV2.S Vaccine. JAMA J. Am. Med. Assoc. 2021 , 325 , 2445–2447. [ Google Scholar ] [ CrossRef ]
• Antrobus, R.D.; Coughlan, L.; Berthoud, T.K.; Dicks, M.D.; Hill, A.V.; Lambe, T.; Gilbert, S.C. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens. Mol. Ther. 2014 , 22 , 668–674. [ Google Scholar ] [ CrossRef ]
• Gregory, A.E.; Titball, R.; Williamson, D. Vaccine delivery using nanoparticles. Front. Cell. Infect. Microbiol. 2013 , 3 , 13. [ Google Scholar ] [ CrossRef ]
• Gheibi Hayat, S.M.; Darroudi, M. Nanovaccine: A novel approach in immunization. J. Cell. Physiol. 2019 , 234 , 12530–12536. [ Google Scholar ] [ CrossRef ]
• Mohsen, M.O.; Zha, L.; Cabral-Miranda, G.; Bachmann, M.F. Major findings and recent advances in virus-like particle (VLP)-based vaccines. Semin. Immunol. 2017 , 34 , 123–132. [ Google Scholar ] [ CrossRef ]
• Marcandalli, J.; Fiala, B.; Ols, S.; Perotti, M.; de van der Schueren, W.; Snijder, J.; Hodge, E.; Benhaim, M.; Ravichandran, R.; Carter, L.; et al. Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus. Cell 2019 , 176 , 1420–1431.e1417. [ Google Scholar ] [ CrossRef ]
• Mallajosyula, J.K.; Hiatt, E.; Hume, S.; Johnson, A.; Jeevan, T.; Chikwamba, R.; Pogue, G.P.; Bratcher, B.; Haydon, H.; Webby, R.J.; et al. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge. Hum. Vaccines Immunother. 2014 , 10 , 586–595. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Steele, J.F.C.; Peyret, H.; Saunders, K.; Castells-Graells, R.; Marsian, J.; Meshcheriakova, Y.; Lomonossoff, G.P. Synthetic plant virology for nanobiotechnology and nanomedicine. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2017 , 9 , e1447. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• McCormick, A.A.; Palmer, K.E. Genetically engineered Tobacco mosaic virus as nanoparticle vaccines. Expert Rev. Vaccines 2008 , 7 , 33–41. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Mansour, A.A.; Banik, S.; Suresh, R.V.; Kaur, H.; Malik, M.; McCormick, A.A.; Bakshi, C.S. An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia. Front. Microbiol. 2018 , 9 , 1195. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Launay, O.; Artaud, C.; Lachatre, M.; Ait-Ahmed, M.; Klein, J.; Luong Nguyen, L.B.; Durier, C.; Jansen, B.; Tomberger, Y.; Jolly, N.; et al. Safety and immunogenicity of a measles-vectored SARS-CoV-2 vaccine candidate, V591/TMV-083, in healthy adults: Results of a randomized, placebo-controlled Phase I study. eBioMedicine 2022 , 75 , 103810. [ Google Scholar ] [ CrossRef ]
• Gao, F.; Weaver, E.A.; Lu, Z.; Li, Y.; Liao, H.X.; Ma, B.; Alam, S.M.; Scearce, R.M.; Sutherland, L.L.; Yu, J.S.; et al. Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein. J. Virol. 2005 , 79 , 1154–1163. [ Google Scholar ] [ CrossRef ]
• Laddy, D.J.; Yan, J.; Corbitt, N.; Kobinger, G.P.; Weiner, D.B. Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine 2007 , 25 , 2984–2989. [ Google Scholar ] [ CrossRef ]
• Santra, S.; Korber, B.T.; Muldoon, M.; Barouch, D.H.; Nabel, G.J.; Gao, F.; Hahn, B.H.; Haynes, B.F.; Letvin, N.L. A centralized gene-based HIV-1 vaccine elicits broad cross-clade cellular immune responses in rhesus monkeys. Proc. Natl. Acad. Sci. USA 2008 , 105 , 10489–10494. [ Google Scholar ] [ CrossRef ]
• Tarr, A.W.; Backx, M.; Hamed, M.R.; Urbanowicz, R.A.; McClure, C.P.; Brown, R.J.P.; Ball, J.K. Immunization with a synthetic consensus hepatitis C virus E2 glycoprotein ectodomain elicits virus-neutralizing antibodies. Antivir. Res. 2018 , 160 , 25–37. [ Google Scholar ] [ CrossRef ]
• Xie, X.; Zhao, C.; He, Q.; Qiu, T.; Yuan, S.; Ding, L.; Liu, L.; Jiang, L.; Wang, J.; Zhang, L.; et al. Influenza Vaccine With Consensus Internal Antigens as Immunogens Provides Cross-Group Protection Against Influenza A Viruses. Front. Microbiol. 2019 , 10 , 1630. [ Google Scholar ] [ CrossRef ]
• Ilyushina, N.A.; Khalenkov, A.M.; Seiler, J.P.; Forrest, H.L.; Bovin, N.V.; Marjuki, H.; Barman, S.; Webster, R.G.; Webby, R.J. Adaptation of pandemic H1N1 influenza viruses in mice. J. Virol. 2010 , 84 , 8607–8616. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Swope, K.; Morton, J.; Pogue, G.P.; Hume, S.; Pauly, M.H.; Shepherd, J.; Simpson, C.A.; Bratcher, B.; Whaley, K.J.; Zeitlin, L.; et al. Manufacturing plant-made monoclonal antibodies for research or therapeutic applications. Methods Enzym. 2021 , 660 , 239–263. [ Google Scholar ] [ CrossRef ]
• Smith, M.L.; Lindbo, J.A.; Dillard-Telm, S.; Brosio, P.M.; Lasnik, A.B.; McCormick, A.A.; Nguyen, L.V.; Palmer, K.E. Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications. Virology 2006 , 348 , 475–488. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hermanson, G.T. Chapter 19—Vaccines and Immunogen Conjugates. In Bioconjugate Techniques , 3rd ed.; Hermanson, G.T., Ed.; Academic Press: Boston, MA, USA, 2013; pp. 839–865. [ Google Scholar ] [ CrossRef ]
• Bullard, B.L.; DeBeauchamp, J.; Pekarek, M.J.; Petro-Turnquist, E.; Vogel, P.; Webby, R.J.; Weaver, E.A. An epitope-optimized human H3N2 influenza vaccine induces broadly protective immunity in mice and ferrets. NPJ Vaccines 2022 , 7 , 65. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Webster, R.G.; Laver, W.G. Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology 1980 , 104 , 139–148. [ Google Scholar ] [ CrossRef ]
• Gerhard, W.; Yewdell, J.; Frankel, M.E.; Webster, R. Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature 1981 , 290 , 713–717. [ Google Scholar ] [ CrossRef ]
• Skehel, J.J.; Stevens, D.J.; Daniels, R.S.; Douglas, A.R.; Knossow, M.; Wilson, I.A.; Wiley, D.C. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc. Natl. Acad. Sci. USA 1984 , 81 , 1779–1783. [ Google Scholar ] [ CrossRef ]
• Goff, P.H.; Krammer, F.; Hai, R.; Seibert, C.W.; Margine, I.; Garcia-Sastre, A.; Palese, P. Induction of cross-reactive antibodies to novel H7N9 influenza virus by recombinant Newcastle disease virus expressing a North American lineage H7 subtype hemagglutinin. J. Virol. 2013 , 87 , 8235–8240. [ Google Scholar ] [ CrossRef ]
• Castrucci, M.R.; Facchini, M.; Di Mario, G.; Garulli, B.; Sciaraffia, E.; Meola, M.; Fabiani, C.; De Marco, M.A.; Cordioli, P.; Siccardi, A.; et al. Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian ‘avian-like’ H1N1 swine viruses in mice. Influenza Other Respir. Viruses 2014 , 8 , 367–375. [ Google Scholar ] [ CrossRef ]
• Liu, S.T.H.; Behzadi, M.A.; Sun, W.; Freyn, A.W.; Liu, W.C.; Broecker, F.; Albrecht, R.A.; Bouvier, N.M.; Simon, V.; Nachbagauer, R.; et al. Antigenic sites in influenza H1 hemagglutinin display species-specific immunodominance. J. Clin. Investig. 2018 , 128 , 4992–4996. [ Google Scholar ] [ CrossRef ]
• Phoolcharoen, W.; Shanmugaraj, B.; Khorattanakulchai, N.; Sunyakumthorn, P.; Pichyangkul, S.; Taepavarapruk, P.; Praserthsee, W.; Malaivijitnond, S.; Manopwisedjaroen, S.; Thitithanyanont, A.; et al. Preclinical evaluation of immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052-Alum adjuvant. Vaccine 2023 , 41 , 2781–2792. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Gasanova, T.V.; Koroleva, A.A.; Skurat, E.V.; Ivanov, P.A. Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization. Biochemistry 2020 , 85 , 224–233. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Pisuttinusart, N.; Shanmugaraj, B.; Srisaowakarn, C.; Ketloy, C.; Prompetchara, E.; Thitithanyanont, A.; Phoolcharoen, W. Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice. Biotechnol. Rep. 2024 , 41 , e00826. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Eidenberger, L.; Kogelmann, B.; Steinkellner, H. Plant-based biopharmaceutical engineering. Nat. Rev. Bioeng. 2023 , 1 , 426–439. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Pogue, G.P.; Vojdani, F.; Palmer, K.E.; Hiatt, E.; Hume, S.; Phelps, J.; Long, L.; Bohorova, N.; Kim, D.; Pauly, M.; et al. Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnol. J. 2010 , 8 , 638–654. [ Google Scholar ] [ CrossRef ]
• Mason, H.S.; Haq, T.A.; Clements, J.D.; Arntzen, C.J. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): Potatoes expressing a synthetic LT-B gene. Vaccine 1998 , 16 , 1336–1343. [ Google Scholar ] [ CrossRef ]
• Mason, H.S.; Lam, D.M.; Arntzen, C.J. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 1992 , 89 , 11745–11749. [ Google Scholar ] [ CrossRef ]
• Yusibov, V.; Shivprasad, S.; Turpen, T.H.; Dawson, W.; Koprowski, H. Plant viral vectors based on tobamoviruses. Curr. Top. Microbiol. Immunol. 1999 , 240 , 81–94. [ Google Scholar ] [ CrossRef ]
• Paul, M.; van Dolleweerd, C.; Drake, P.M.; Reljic, R.; Thangaraj, H.; Barbi, T.; Stylianou, E.; Pepponi, I.; Both, L.; Hehle, V.; et al. Molecular Pharming: Future targets and aspirations. Hum. Vaccin. 2011 , 7 , 375–382. [ Google Scholar ] [ CrossRef ]
• Klimyuk, V.; Pogue, G.; Herz, S.; Butler, J.; Haydon, H. Production of recombinant antigens and antibodies in Nicotiana benthamiana using ‘magnifection’ technology: GMP-compliant facilities for small- and large-scale manufacturing. Curr. Top. Microbiol. Immunol. 2014 , 375 , 127–154. [ Google Scholar ] [ CrossRef ]
• Chen, M.; Liu, X.; Wang, Z.; Song, J.; Qi, Q.; Wang, P.G. Modification of plant N-glycans processing: The future of producing therapeutic protein by transgenic plants. Med. Res. Rev. 2005 , 25 , 343–360. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Ducatez, M.F.; Bahl, J.; Griffin, Y.; Stigger-Rosser, E.; Franks, J.; Barman, S.; Vijaykrishna, D.; Webb, A.; Guan, Y.; Webster, R.G.; et al. Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development. Proc. Natl. Acad. Sci. USA 2011 , 108 , 349–354. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Sumida, S.M.; Truitt, D.M.; Lemckert, A.A.; Vogels, R.; Custers, J.H.; Addo, M.M.; Lockman, S.; Peter, T.; Peyerl, F.W.; Kishko, M.G.; et al. Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein. J. Immunol. 2005 , 174 , 7179–7185. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Barouch, D.H.; Pau, M.G.; Custers, J.H.; Koudstaal, W.; Kostense, S.; Havenga, M.J.; Truitt, D.M.; Sumida, S.M.; Kishko, M.G.; Arthur, J.C.; et al. Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity. J. Immunol. 2004 , 172 , 6290–6297. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Valkenburg, S.A.; Mallajosyula, V.V.; Li, O.T.; Chin, A.W.; Carnell, G.; Temperton, N.; Varadarajan, R.; Poon, L.L. Stalking influenza by vaccination with pre-fusion headless HA mini-stem. Sci. Rep. 2016 , 6 , 22666. [ Google Scholar ] [ CrossRef ]
• Coudeville, L.; Bailleux, F.; Riche, B.; Megas, F.; Andre, P.; Ecochard, R. Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: Development and application of a bayesian random-effects model. BMC Med. Res. Methodol. 2010 , 10 , 18. [ Google Scholar ] [ CrossRef ]
• Black, S.; Nicolay, U.; Vesikari, T.; Knuf, M.; Del Giudice, G.; Della Cioppa, G.; Tsai, T.; Clemens, R.; Rappuoli, R. Hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children. Pediatr. Infect. Dis. J. 2011 , 30 , 1081–1085. [ Google Scholar ] [ CrossRef ]
• Hobson, D.; Curry, R.L.; Beare, A.S.; Ward-Gardner, A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. Epidemiol. Infect. 1972 , 70 , 767–777. [ Google Scholar ] [ CrossRef ]
• Kaufmann, L.; Syedbasha, M.; Vogt, D.; Hollenstein, Y.; Hartmann, J.; Linnik, J.E.; Egli, A. An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers. J. Vis. Exp. 2017 , 130 , e55833. [ Google Scholar ] [ CrossRef ]
• Bibby, D.C.; Savanovic, M.; Zhang, J.; Torelli, A.; Jeeninga, R.E.; Gagnon, L.; Harris, S.L. Interlaboratory Reproducibility of Standardized Hemagglutination Inhibition Assays. mSphere 2022 , 7 , e0095321. [ Google Scholar ] [ CrossRef ]
• Zhou, F.; Hansen, L.; Pedersen, G.; Grodeland, G.; Cox, R. Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection. Front. Immunol. 2021 , 12 , 747774. [ Google Scholar ] [ CrossRef ]
• Monto, A.S.; Petrie, J.G.; Cross, R.T.; Johnson, E.; Liu, M.; Zhong, W.; Levine, M.; Katz, J.M.; Ohmit, S.E. Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection. J. Infect. Dis. 2015 , 212 , 1191–1199. [ Google Scholar ] [ CrossRef ]
• Doud, M.B.; Lee, J.M.; Bloom, J.D. How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin. Nat. Commun. 2018 , 9 , 1386. [ Google Scholar ] [ CrossRef ]
• Kilbourne, E.D.; Smith, C.; Brett, I.; Pokorny, B.A.; Johansson, B.; Cox, N. The total influenza vaccine failure of 1947 revisited: Major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic. Proc. Natl. Acad. Sci. USA 2002 , 99 , 10748–10752. [ Google Scholar ] [ CrossRef ]
• Kodihalli, S.; Justewicz, D.M.; Gubareva, L.V.; Webster, R.G. Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective. J. Virol. 1995 , 69 , 4888–4897. [ Google Scholar ] [ CrossRef ]
• Ray, R.; Nait Mohamed, F.A.; Maurer, D.P.; Huang, J.; Alpay, B.A.; Ronsard, L.; Xie, Z.; Han, J.; Fernandez-Quintero, M.; Phan, Q.A.; et al. Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses. Immunity 2024 , 57 , 1141–1159.e11. [ Google Scholar ] [ CrossRef ]

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Madapong, A.; Petro-Turnquist, E.M.; Webby, R.J.; McCormick, A.A.; Weaver, E.A. Immunity and Protective Efficacy of a Plant-Based Tobacco Mosaic Virus-like Nanoparticle Vaccine against Influenza a Virus in Mice. Vaccines 2024 , 12 , 1100. https://doi.org/10.3390/vaccines12101100

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Advancements in dsRNA-based approaches: a comprehensive review on potent strategies for plant disease management

• Review Article
• Published: 24 September 2024

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• D. S. Srimahesvari 1 ,
• S. Harish   ORCID: orcid.org/0000-0001-5703-9887 1 ,
• G. Karthikeyan 1 ,
• M. Kannan 2 &
• K. K. Kumar 3  

Plant diseases constitute notable threats to modern agriculture and managing them is a major challenge during crop cultivation. Plant disease management has long been one of the primary goals of any crop development program. Various traditional approaches used by the farming community such as pesticides have raised major concerns for the environment and human health. Among the biotechnological approaches used during the last two decades, RNA silencing-based resistance has been considered an important strategy for developing resistant crops. Engineered plants particularly those expressing RNA-silencing nucleotides are becoming increasingly relevant and are expected to deliver more effective solutions. Recent studies have shown that exogenous application of dsRNA targeting fungal growth and pathogenicity-related genes facilitates the reduction of pathogen and symptom expression. These findings indicate that topical application of dsRNA holds great potential in plant disease management. In this review, we explain the RNAi mechanism, methods involved in dsRNA synthesis, uptake efficiency, stability enhancement and its application. Besides, it also highlights the role of dsRNA in plant disease management and factors that influence its efficiency to offer appropriate measures.

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Topical application of dsRNA for plant virus control: a review

Host-Induced Gene Silencing: Approaches in Plant Disease Management

The future is now: revolution of RNA-mediated gene silencing in plant protection against insect pests and diseases

Abbreviations.

Ribonucleic acid

Double-stranded RNA

Transposable elements

Last Eukaryotic Common Ancestor

Spray-induced gene silencing

Gene silencing

Piwi Argonaute Zwille

Double-stranded RNA binding site

RNA dependent RNA polymerase

RNA induced silencing complex

Post-transcriptional gene silencing

Transcriptional gene silencing

Short interfering RNA

Messenger RNA

RNA dependent DNA methylation

Multiple cloning sites

Isopropyl-β-D- thiogalactopyranoside

Pepper mild mottle virus

Potato virus Y

Tobacco mosaic virus

Sugarcane mosaic virus

Zucchini yellow mosaic virus

Cucumber mosaic virus

Tomato leaf curl virus

Bean common mosaic virus

Tomato mosaic virus

Tomato spotted wilt virus

Papaya ring spot virus

Pigeonpea sterility mosaic virus

Sesbania mosaic virus

Mungbean yellow mosaic virus

Citrus tristeza virus

Potato virus X

Tomato Leaf Curl New Delhi Virus

Coat Protein

Movement Protein

Nucleoprotein

Cytochrome P450

Modulator of Apoptosis 1

Vacuolar protein sorting

Chalcone synthase

Transcription-activator-like effectors

Salt- and drought-induced RING box

Phytoene desaturase

Xanthomonas oryzae pv. oryzae

Tryptophan-2- monooxygenase & Isopentyl transferase

HrpN interacting protein from malus

Host-induced gene silencing

1-Aminocyclopropane-1-carboxylate

Artificial microRNA

TransactingRNA

ExogenicsiRNA

Environmental Protection Agency

Genetically Modified Organisms

Domain-rearranged methyltransferase 2

Methyltransferase 1

Chromo methylase 3

Layered Double Hydroxides

Chitosan nanoparticles

Star polycations

Paperclip RNA

Clathrin-mediated endocytosis

Polyethyleneimine

Single-walled carbon nanotubes

Cell-penetrating peptide

Graphene Quantum dots

Graphene Quantum Dots- branched polyethlenimine

Ultraviolet Light amplification by stimulated emission of radiation

Double-stranded proteasome subunit beta type 5

Aalto AP, Sarin LP, Van Dijk AA, Saarma M, Poranen MM, Arumäe U et al (2007) Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase. RNA 13(3):422–429

Article   PubMed   PubMed Central   CAS   Google Scholar  

Abbasi Khalaki M, Moameri M, Asgari Lajayer B, Astatkie T (2021) Influence of nano-priming on seed germination and plant growth of forage and medicinal plants. Plant Growth Regul 93(1):13–28

Article   CAS   Google Scholar  

Ahn SJ, Donahue K, Koh Y, Martin RR, Choi MY (2019) Microbial-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management. Int J Insect Sci 11:1179543319840323

Article   PubMed   PubMed Central   Google Scholar  

Alamalakala L, Parimi S, Patel N, Char B (2018) Insect RNAi: integrating a new tool in the crop protection toolkit. Trends Insect Mol Biol Biotechnol 193–232

Arias RS, Dang PM, Sobolev VS (2015) RNAi-mediated control of aflatoxins in peanut: method to analyze mycotoxin production and transgene expression in the peanut/Aspergillus pathosystem. JoVE J vis Exp 106:e53398

Google Scholar  

Arora AK, Chung SH, Douglas AE (2021) Non-target effects of dsRNA molecules in hemipteran insects. Genes 12(3):407

Azizi A, del Río Mendoza LE (2024) Effective control of Sclerotinia stem rot in canola plants through application of exogenous hairpin RNA of multiple Sclerotinia sclerotiorum genes. Phytopathology 114(5):1000–1010

Article   PubMed   CAS   Google Scholar  

Bachman PM, Huizinga KM, Jensen PD, Mueller G, Tan J, Uffman JP et al (2016) Ecological risk assessment for DvSnf7 RNA: a plant-incorporated protectant with targeted activity against western corn rootworm. Regul Toxicol Pharmacol 81:77–88

Bachman P, Fischer J, Song Z, Urbanczyk-Wochniak E, Watson G (2020) Environmental fate and dissipation of applied dsRNA in soil, aquatic systems, and plants. Front Plant Sci 11:21

Bakade R, Ingole KD, Deshpande S, Pal G, Patil SS, Bhattacharjee S et al (2021) Comparative transcriptome analysis of rice resistant and susceptible genotypes to Xanthomonas oryzae pv. oryzae identifies novel genes to control bacterial leaf blight. Mol Biotechnol 63(8):719–731

Basso MF, Neves MF, Grossi-de-Sa MF (2024) Agriculture evolution, sustainability and trends, focusing on Brazilian agribusiness: a review. Front Sustain Food Syst 7:1296337

Article   Google Scholar  

Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O et al (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25(11):1322–1326

Bauman J, Jearawiriyapaisarn N, Kole R (2009) Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides 19(1):1–13

Bennur SV, Ram M (2024) Marker-assisted breeding techniques for the development of gluten-free wheat varieties: a comprehensive review. J Adv Biol Biotechnol 27(5):256–267

Bezrutczyk M, Yang J, Eom JS, Prior M, Sosso D, Hartwig T et al (2018) Sugar flux and signaling in plant–microbe interactions. Plant J 93(4):675–685

Bharathi JK, Anandan R, Benjamin LK, Muneer S, Prakash MAS (2023) Recent trends and advances of RNA interference (RNAi) to improve agricultural crops and enhance their resilience to biotic and abiotic stresses. Plant Physiol Biochem 194:600–618

Bocos-Asenjo IT, Amin H, Mosquera S, Díez-Hermano S, Ginesy M, Diez JJ, et al (2024) Spray-induced gene silencing (SIGS) as a tool for the management of Pine Pitch Canker forest disease. bioRxiv 2024.03. 05.583474

Borah M, Nath PD, Chaudhury SP, Biswas KK, Patil BL, Voloudakis A (2024) Topical application of dsRNAs targeting Citrus tristeza virus (CTV) reduces its titer in the CTV infected sweet orange (Citrus sinensis). Eur J Plant Pathol 168(2):273–278

Bragg Z, Rieske LK (2022) Feasibility of systemically applied dsRNAs for pest-specific RNAi-induced gene silencing in white oak. Front Plant Sci 13:830226

Carbonell A, de Alba ÁEM, Flores R, Gago S (2008) Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families. Virology 371(1):44–53

Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N et al (2005) The transcriptional landscape of the mammalian genome. Science 309(5740):1559–1563

Cedillo-Jimenez CA, Guevara-Gonzalez RG, Cruz-Hernandez A (2024) Exogenous dsRNA sequence based on miR1917 downregulates its target gene related to ethylene signaling in tomato seedlings and fruit. Sci Hortic 331:113090

Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8(11):884–896

Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18(10):1179–1186

Chen Y, De Schutter K (2024) Biosafety aspects of RNAi-based pests control. Pest Manag Sci. https://doi.org/10.1002/ps.8098

Article   PubMed   Google Scholar  

Chilakamarthi U, Mukherjee SK, Deb JK (2007) Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein. FEBS Lett 581(14):2675–2683

Choudhary S, Thakur S, Bhardwaj P (2019) Molecular basis of transitivity in plant RNA silencing. Mol Biol Rep 46(4):4645–4660

Christiaens O, Dzhambazova T, Kostov K, Arpaia S, Joga MR, Urru I et al (2018) Literature review of baseline information on RNAi to support the environmental risk assessment of RNAi-based GM plants. EFSA Support Publ 15(5):1424E

Dalakouras A, Jarausch W, Buchholz G, Bassler A, Braun M, Manthey T et al (2018) Delivery of hairpin RNAs and small RNAs into woody and herbaceous plants by trunk injection and petiole absorption. Front Plant Sci 9:1253

Dalakouras A, Koidou V, Papadopoulou K (2024) DsRNA-based pesticides: Considerations for efficiency and risk assessment. Chemosphere 141530

Damalas CA, Eleftherohorinos IG (2011) Pesticide exposure, safety issues, and risk assessment indicators. Int J Environ Res Public Health 8(5):1402–1419

Das PR, Sherif SM (2020) Application of exogenous dsRNAs-induced RNAi in agriculture: challenges and triumphs. Front Plant Sci 11:946

Davuluri GR, Van Tuinen A, Fraser PD, Manfredonia A, Newman R, Burgess D et al (2005) Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol 23(7):890–895

Degnan RM, Shuey LS, Radford-Smith J, Gardiner DM, Carroll BJ, Mitter N et al (2023) Double-stranded RNA prevents and cures infection by rust fungi. Commun Biol 6(1):1234

Delteil A, Gobbato E, Cayrol B, Estevan J, Michel-Romiti C, Dievart A et al (2016) Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus. BMC Plant Biol 16:1–10

Demilie WB (2024) Plant disease detection and classification techniques: a comparative study of the performances. J Big Data 11(1):5

Demirer GS, Silva TN, Jackson CT, Thomas JB, Ehrhardt DW, Rhee SY, Mortimer JC, Landry MP (2021) Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nat Nanotechnol 16(3):243–250

Deutsch CA, Tewksbury JJ, Tigchelaar M, Battisti DS, Merrill SC, Huey RB et al (2018) Increase in crop losses to insect pests in a warming climate. Science 361(6405):916–919

Dubelman S, Fischer J, Zapata F, Huizinga K, Jiang C, Uffman J et al (2014) Environmental fate of double-stranded RNA in agricultural soils. PLoS ONE 9(3):e93155

Dubrovina AS, Kiselev KV (2019) Exogenous RNAs for gene regulation and plant resistance. Int J Mol Sci 20(9):2282

Dubrovina AS, Aleynova OA, Suprun AR, Ogneva ZV, Kiselev KV (2020) Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs. Appl Microbiol Biotechnol 104:2125–2135

Dunoyer P, Himber C, Ruiz-Ferrer V, Alioua A, Voinnet O (2007) Intra-and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat Genet 39(7):848–856

Escobar MA, Dandekar AM (2003) Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci 8(8):380–386

Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc Natl Acad Sci 98(23):13437–13442

Ferguson CM, Echeverria D, Hassler M, Ly S, Khvorova A (2020) Cell type impacts accessibility of mRNA to silencing by RNA interference. Mol Ther-Nucleic Acids 21:384–393

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811

Fischer JR, Zapata F, Dubelman S, Mueller GM, Uffman JP, Jiang C et al (2017) Aquatic fate of a double-stranded RNA in a sediment–water system following an over-water application. Environ Toxicol Chem 36(3):727–734

Frascati F, Rotunno S, Accotto GP, Noris E, Vaira AM, Miozzi L (2024) Exogenous application of dsRNA for Protection against tomato leaf curl new Delhi virus. Viruses 16(3):436

Fukusaki EI, Kawasaki K, Kajiyama SI, An CI, Suzuki K, Tanaka Y, Kobayashi A (2004) Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference. J Biotechnol 111(3):229–240

Gaffar FY, Koch A (2019) Catch me if you can! RNA silencing-based improvement of antiviral plant immunity. Viruses 11(7):673

Gan D, Zhang J, Jiang H, Jiang T, Zhu S, Cheng B (2010) Bacterially expressed dsRNA protects maize against SCMV infection. Plant Cell Rep 29:1261–1268

Gebremichael DE, Haile ZM, Negrini F, Sabbadini S, Capriotti L, Mezzetti B et al (2021) RNA interference strategies for future management of plant pathogenic fungi: prospects and challenges. Plants 10(4):650

Ghag SB (2021) RNAi strategy for management of phytopathogenic fungi. Elsevier, CRISPR and RNAi Systems

Book   Google Scholar  

Ghag SB, Alok A, Rajam MV, Penna S (2023) Designing climate-resilient crops for sustainable agriculture: a silent approach. J Plant Growth Regul 42(10):6503–6522

Ghosh SKB, Gundersen-Rindal DE (2017) Double strand RNA-mediated RNA interference through feeding in larval gypsy moth, Lymantria dispar (Lepidoptera: Erebidae). Eur J Entomol 114:170–178

Gilbert MK, Majumdar R, Rajasekaran K, Chen ZY, Wei Q, Sickler CM et al (2018) RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels. Planta 247:1465–1473

Good L, Stach JE (2011) Synthetic RNA silencing in bacteria–antimicrobial discovery and resistance breaking. Front Microbiol 2:185

Gordon DM (2018) Reading the archives as sources. In: Oxford Research Encyclopedia of African History

Goulin EH, Galdeano DM, Granato LM, Matsumura EE, Dalio RJD, Machado MA (2019) RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens. Microbiol Res 226:1–9

Govindarajulu M, Epstein L, Wroblewski T, Michelmore RW (2015) Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Plant Biotechnol J 13(7):875–883

Guo XY, Li Y, Fan J, Xiong H, Xu FX, Shi J et al (2019) Host-induced gene silencing of MoAP1 confers broad-spectrum resistance to Magnaporthe oryzae. Front Plant Sci 10:433

Gyawali B, Rahimi R, Alizadeh H, Mohammadi M (2024) Graphene quantum dots (GQD)-mediated dsRNA delivery for the control of fusarium head blight disease in wheat. ACS Appl Bio Mater 7(3):1526–1535

Hannon GJ (2002) RNA interference. Nature 418(6894):244–251

Hao Z, Wang M, Cheng L, Si M, Feng Z, Feng Z (2024) Synergistic antibacterial mechanism of silver-copper bimetallic nanoparticles. Front Bioeng Biotechnol 11:1337543

Hariharan G, Sivasubramaniam N, Prasannath K (2021) RNA interference as a promising strategy for plant disease management. Elsevier, Food Security and Plant Disease Management

Hashiro S, Mitsuhashi M, Chikami Y, Kawaguchi H, Niimi T, Yasueda H (2019) Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control. Appl Microbiol Biotechnol 103(20):8485–8496

Heinemann JA, Agapito-Tenfen SZ, Carman JA (2013) A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environ Int 55:43–55

Holeva MC, Sklavounos A, Rajeswaran R, Pooggin MM, Voloudakis AE (2021) Topical application of double-stranded RNA targeting 2b and CP genes of Cucumber mosaic virus protects plants against local and systemic viral infection. Plants 10(5):963

Hu Z, Guo J, Da Gao B, Zhong J (2020) A novel mycovirus isolated from the plant-pathogenic fungus Alternaria dianthicola. Arch Virol 165:2105–2109

Huvenne H, Smagghe G (2010) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J Insect Physiol 56(3):227–235

Irie A, Sato K, Hara RI, Wada T, Shibasaki F (2020) An artificial cationic oligosaccharide combined with phosphorothioate linkages strongly improves siRNA stability. Sci Rep 10(1):14845

Islam MT, Davis Z, Chen L, Englaender J, Zomorodi S, Frank J et al (2021) Minicell-based fungal RNAi delivery for sustainable crop protection. Microb Biotechnol 14(4):1847–1856

Jiang YX, Chen JZ, Li MW, Zha BH, Huang PR, Chu XM et al (2021) The combination of Bacillus thuringiensis and its engineered strain expressing dsRNA increases the toxicity against Plutella xylostella. Int J Mol Sci 23(1):444

Joga MR, Zotti MJ, Smagghe G, Christiaens O (2016) RNAi efficiency, systemic properties, and novel delivery methods for pest insect control: what we know so far. Front Physiol 7:553

Jones RC, Koenig R, Lesemann DE (1980) Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum). Ann Appl Biol 94(1):61–68

Kabi M, Tripathy SK, Dash M, Baisakh B, Das TR, Kesawat MS (2024) Reverse breeding: a novel tool for crop improvement. Apple Academic Press, Smart Breeding

Kaldis A, Berbati M, Melita O, Reppa C, Holeva M, Otten P et al (2018) Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV. Mol Plant Pathol 19(4):883–895

Kamesh Krishnamoorthy K, Malathi VG, Renukadevi P, Kumar SM, Raveendran M, Sudha M et al (2023) Exogenous delivery of dsRNA for management of mungbean yellow mosaic virus on blackgram. Planta 258(5):94

Kamthan A, Chaudhuri A, Kamthan M, Datta A (2015) Small RNAs in plants: recent development and application for crop improvement. Front Plant Sci 6:208

Kaur L, Sohal HS, Kaur M, Malhi DS, Garg S (2020) A mini-review on nano technology in the tumour targeting strategies: drug delivery to cancer cells. Anti-Cancer Agents Med Chem Former Curr Med Chem-Anti-Cancer Agents 20(17):2012–2024

CAS   Google Scholar  

Kettles GJ, Hofinger BJ, Hu P, Bayon C, Rudd JJ, Balmer D et al (2019) sRNA profiling combined with gene function analysis reveals a lack of evidence for cross-kingdom RNAi in the wheat–Zymoseptoria tritici pathosystem. Front Plant Sci 10:892

Kim S, Lee R, Jeon H, Lee N, Park J, Moon H et al (2023) Identification of essential genes for the establishment of spray-induced gene silencing-based disease control in Fusarium graminearum. J Agric Food Chem 71(49):19302–19311

Kiselev KV, Suprun AR, Aleynova OA, Ogneva ZV, Kostetsky EY, Dubrovina AS (2022) The specificity of transgene suppression in plants by exogenous dsRNA. Plants 11(6):715

Koch A, Kogel KH (2014) New wind in the sails: improving the agronomic value of crop plants through RNA i-mediated gene silencing. Plant Biotechnol J12(7):821–831

Koch A, Biedenkopf D, Furch A, Weber L, Rossbach O, Abdellatef E et al (2016) An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathog 12(10):e1005901

Kolge H, Kadam K, Galande S, Lanjekar V, Ghormade V (2021) New frontiers in pest control: chitosan nanoparticles-shielded dsRNA as an effective topical RNAi spray for gram podborer biocontrol. ACS Appl Bio Mater 4(6):5145–5157

Konakalla NC, Bag S, Deraniyagala AS, Culbreath AK, Pappu HR (2021) Induction of plant resistance in tobacco (Nicotiana tabacum) against tomato spotted wilt orthotospovirus through foliar application of dsRNA. Viruses 13(4):662

Kujur NMJ, Doggalli G, Solanki RS, Saran D, Dhanalakshmi TN (2024) Revolutionizing Field Crop Breeding

Kulkarni MM, Booker M, Silver SJ, Friedman A, Hong P, Perrimon N et al (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat Methods 3(10):833–838

Lai CK, Miller MC, Collins K (2003) Roles for RNA in telomerase nucleotide and repeat addition processivity. Mol Cell 11(6):1673–1683

Ledger SE, Janssen BJ, Karunairetnam S, Wang T, Snowden KC (2010) Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis). New Phytol 188(3):803–813

Levanova AA, Poranen MM (2024) Utilization of bacteriophage phi6 for the production of high-quality double-stranded RNA molecules. Viruses 16(1):166

Lindbo JA (2012) A historical overview of RNAi in plants. Antivir Resist Plants Methods Protoc. https://doi.org/10.1007/978-1-61779-882-5_1

Luo Y, Chen Q, Luan J, Chung SH, Van Eck J, Turgeon R et al (2017) Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci. Insect Biochem Mol Biol 88:21–29

Ma Z, Zhou H, Wei Y, Yan S, Shen J (2020) A novel plasmid– Escherichia coli system produces large batch dsRNAs for insect gene silencing. Pest Manag Sci 76(7):2505–2512

Mahto BK, Singh A, Pareek M, Rajam MV, Dhar-Ray S, Reddy PM (2020) Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato. Plant Mol Biol 104:381–395

Malnoy M, Jin Q, Borejsza-Wysocka EE, He SY, Aldwinckle HS (2007) Overexpression of the apple MpNPR1 gene confers increased disease resistance in Malus×domestica. Mol Plant Microbe Interact 20(12):1568–1580

Mamta B, Rajam MV (2017) RNAi technology: a new platform for crop pest control. Physiol Mol Biol Plants 23:487–501

Mann SK, Kashyap PL, Sanghera GS, Singh G, Singh S (2008) RNA interference: an eco-friendly tool for plant disease management. Transgenic Plant J 2(2):110–126

McLoughlin AG, Wytinck N, Walker PL, Girard IJ, Rashid KY, de Kievit T et al (2018) Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea. Sci Rep 8(1):7320

Meli VS, Ghosh S, Prabha TN, Chakraborty N, Chakraborty S, Datta A (2010) Enhancement of fruit shelf life by suppressing N-glycan processing enzymes. Proc Natl Acad Sci 107(6):2413–2418

Melita O, Kaldis A, Berbati M, Reppa C, Holeva M, Lapidot M et al (2021) Topical application of double-stranded RNA molecules deriving from tomato yellow leaf curl virus reduces cognate virus infection in tomato. Biol Plant 65(1):100–110

Mellbye BL, Puckett SE, Tilley LD, Iversen PL, Geller BL (2009) Variations in amino acid composition of antisense peptide-phosphorodiamidate morpholino oligomer affect potency against Escherichia coli in vitro and in vivo. Antimicrob Agents Chemother 53(2):525–530

Meng L, Hua F, Bian Z (2020) Coronavirus disease 2019 (COVID-19): emerging and future challenges for dental and oral medicine. J Dent Res 99(5):481–487

Mensink BH (2008) How to evaluate the environmental safety of microbial crop protection products? A proposal. Lab FIELD–KEY POINTS 31:28–31

Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C et al (2017) Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants 3(2):1–10

Mosa MA, Youssef K (2021) Topical delivery of host induced RNAi silencing by layered double hydroxide nanosheets: an efficient tool to decipher pathogenicity gene function of Fusarium crown and root rot in tomato. Physiol Mol Plant Pathol 115:101684

Mumbanza FM, Kiggundu A, Tusiime G, Tushemereirwe WK, Niblett C, Bailey A (2013) In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis. Pest Manag Sci 69(10):1155–1162

Murphy KA, Tabuloc CA, Cervantes KR, Chiu JC (2016) Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci Rep 6(1):22587

Namgial T, Kaldis A, Chakraborty S, Voloudakis A (2019) Topical application of double-stranded RNA molecules containing sequences of Tomato leaf curl virus and Cucumber mosaic virus confers protection against the cognate viruses. Physiol Mol Plant Pathol 108:101432

Necira K, Contreras L, Kamargiakis S, Kamoun MS, Canto T, Tenllado F (2024) Comparative analysis of RNA interference and pattern-triggered immunity induced by dsRNA reveals different efficiencies in the antiviral response to Potato virus X. bioRxiv 2024.02. 07.579064

Nerva L, Sandrini M, Gambino G, Chitarra W (2020) Double-stranded RNAs (dsRNAs) as a sustainable tool against gray mold (Botrytis cinerea) in grapevine: effectiveness of different application methods in an open-air environment. Biomolecules 10(2):200

Nicolopoulou-Stamati P, Maipas S, Kotampasi C, Stamatis P, Hens L (2016) Chemical pesticides and human health: the urgent need for a new concept in agriculture. Front Public Health 4:148

Niehl A, Soininen M, Poranen MM, Heinlein M (2018) Synthetic biology approach for plant protection using dsRNA. Plant Biotechnol J 16(9):1679–1687

Niu D, Hamby R, Sanchez JN, Cai Q, Yan Q, Jin H (2021) RNAs: a new frontier in crop protection. Curr Opin Biotechnol 70:204–212

Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D et al (2010) HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22(9):3130–3141

Oliva R, Ji C, Atienza-Grande G, Huguet-Tapia JC, Perez-Quintero A, Li T et al (2019) Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol 37(11):1344–1350

Padmanaban P, Raman PS, Mavalankar DV (2009) Innovations and challenges in reducing maternal mortality in Tamil Nadu. India J Health Popul Nutr 27(2):202

PubMed   CAS   Google Scholar  

Pal S, Dam S (2022) CRISPR-Cas9: taming protozoan parasites with bacterial scissor. J Parasit Dis 46(4):1204–1212

Park M, Um TY, Jang G, Choi YD, Shin C (2022) Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana. Appl Biol Chem 65(1):4

Patil BL, Fauquet CM (2015) Studies on differential behavior of cassava mosaic geminivirus DNA components, symptom recovery patterns, and their siRNA profiles. Virus Genes 50(3):474–486

Patil BL, Raghu R, Dangwal M, Byregowda M, Voloudakis A (2021) Exogenous dsRNA-mediated field protection against Pigeonpea sterility mosaic emaravirus. J Plant Biochem Biotechnol 30:400–405

Patwa N, Gupta OP, Pandey V, Yadav A (2023) RNAi based approaches for abiotic and biotic stresses tolerance of crops. In: Plant Small RNA in Food Crops. Elsevier 183–214

Peláez P, Sanchez F (2013) Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences. Front Plant Sci 4:343

Puyam A, Sharma S, Kashyap PL (2017) RNA interference-a novel approach for plant disease management. J Appl Nat Sci 9(3):1612–1618

Qi T, Zhu X, Tan C, Liu P, Guo J, Kang Z et al (2018) Host-induced gene silencing of an important pathogenicity factor P s CPK 1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust. Plant Biotechnol J 16(3):797–807

Qiao L, Lan C, Capriotti L, Ah-Fong A, Nino Sanchez J, Hamby R et al (2021) Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol J 19(9):1756–1768

Rafique A, Afroz A, Zeeshan N, Rashid U, Khan MAU, Irfan M et al (2023) Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray. PLoS ONE 18(5):e0284888

Rank AP, Koch A (2021) Lab-to-field transition of RNA spray applications–how far are we? Front Plant Sci 12:755203

Raybould A (2006) Problem formulation and hypothesis testing for environmental risk assessments of genetically modified crops. Environ Biosafety Res 5(3):119–125

Raybould A, Burns A (2020) Problem formulation for off-target effects of externally applied double-stranded RNA-based products for pest control. Front Plant Sci 11:424

Rego-Machado CM, Nakasu EY, Silva JM, Lucinda N, Nagata T, Inoue-Nagata AK (2020) siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants. Sci Rep 10(1):22277

Sang H, Kim JI (2020) Advanced strategies to control plant pathogenic fungi by host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS). Plant Biotechnol Rep 14(1):1–8

Saurabh S, Vidyarthi AS, Prasad D (2014) RNA interference: concept to reality in crop improvement. Planta 239:543–564

Sauve-Ciencewicki A, Davis KP, McDonald J, Ramanarayanan T, Raybould A, Wolf DC et al (2019) A simple problem formulation framework to create the right solution to the right problem. Regul Toxicol Pharmacol 101:187–193

Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A (2019) The global burden of pathogens and pests on major food crops. Nat Ecol Evol 3(3):430–439

De Schutter K, Taning CNT, Van Daele L, Van Damme EJ, Dubruel P, Smagghe G (2022) RNAi-based biocontrol products: Market status, regulatory aspects, and risk assessment. Vol. 1, Frontiers in Insect Science. Frontiers Media SA

Schwartz SH, Hendrix B, Hoffer P, Sanders RA, Zheng W (2020) Carbon dots for efficient small interfering RNA delivery and gene silencing in plants. Plant Physiol 184(2):647–657

Shabalina SA, Spiridonov NA (2004) The mammalian transcriptome and the function of non-coding DNA sequences. Genome Biol 5:1–8

Sharma S, Kumar A, Kumari N, Walia A (2023) RNAi as a tool to enhance crop yield and biotic stress management in the plants. Plant Cell Tissue Organ Cult PCTOC 152(3):437–454

Sherman JH, Munyikwa T, Chan SY, Petrick JS, Witwer KW, Choudhuri S (2015) RNAi technologies in agricultural biotechnology: the toxicology forum 40th annual summer meeting. Regul Toxicol Pharmacol 73(2):671–680

Singewar K, Fladung M (2023) Double-stranded RNA (dsRNA) technology to control forest insect pests and fungal pathogens: challenges and opportunities. Funct Integr Genomics 23(2):185

Singh RK, Krishnamachari A, Sharaff M (2020) Challenges of small RNA technology. Elsevier, Plant Small RNA

Singh A, Roychoudhury A (2023) RNA interference (RNAi) technology: an effective tool in plant breeding. In: Advanced Crop Improvement, Volume 1: Theory and Practice. Springer International Publishing Cham 309–20

Song XS, Gu KX, Duan XX, Xiao XM, Hou YP, Duan YB et al (2018) Secondary amplification of siRNA machinery limits the application of spray-induced gene silencing. Mol Plant Pathol 19(12):2543–2560

Sun ZN, Yin GH, Song YZ, An HL, Zhu CX, Wen FJ (2010) Bacterially expressed double-stranded RNAs against hot-spot sequences of tobacco mosaic virus or potato virus Y genome have different ability to protect tobacco from viral infection. Appl Biochem Biotechnol 162:1901–1914

Szittya G, Silhavy D, Molnár A, Havelda Z, Lovas Á, Lakatos L et al (2003) Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 22:633–640

Tabein S, Jansen M, Noris E, Vaira AM, Marian D, Behjatnia SAA et al (2020) The induction of an effective dsRNA-mediated resistance against tomato spotted wilt virus by exogenous application of double-stranded RNA largely depends on the selection of the viral RNA target region. Front Plant Sci 11:533338

Takiff HE, Chen SM, Court DL (1989) Genetic analysis of the rnc operon of Escherichia coli. J Bacteriol 171(5):2581–2590

Tenllado F, Dıaz-Ruız JR (2001) Double-stranded RNA-mediated interference with plant virus infection. J Virol 75(24):12288–12297

Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR (2003) Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. Bmc Biotechnol 3:1–11

Thagun C, Horii Y, Mori M, Fujita S, Ohtani M, Tsuchiya K et al (2022) Non-transgenic gene modulation via spray delivery of nucleic acid/peptide complexes into plant nuclei and chloroplasts. ACS Nano 16(3):3506–3521

Thakre N, Carver M, Paredes-Montero JR, Mondal M, Hu J, Saberi E et al (2024) UV-LASER adjuvant–surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid. Pest Manag Sci 80(4):2141–2153

Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263(1–2):103–112

Tiwari IM, Jesuraj A, Kamboj R, Devanna BN, Botella JR, Sharma TR (2017) Host delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani). Sci Rep 7(1):7521

Torri A, Jaeger J, Pradeu T, Saleh MC (2022) The origin of RNA interference: adaptive or neutral evolution? PLoS Biol 20(6):e3001715

Tretiakova P, Voegele RT, Soloviev A, Link TI (2022) Successful silencing of the mycotoxin synthesis gene TRI5 in Fusarium culmorum and observation of reduced virulence in VIGS and SIGS experiments. Genes 13(3):395

Tricoll DM, Carney KJ, Russell PF, McMaster JR, Groff DW, Hadden KC et al (1995) Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Bio/technology 13(12):1458–1465

Vadlamudi T, Patil BL, Kaldis A, Gopal DVRS, Mishra R, Berbati M et al (2020) DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya. J Virol Methods 275:113750

Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114(17):3083–3091

Vemanna RS, Bakade R, Bharti P, Kumar MP, Sreeman SM, Senthil-Kumar M et al (2019) Cross-talk signaling in rice during combined drought and bacterial blight stress. Front Plant Sci 10:193

Vetukuri RR, Dubey M, Kalyandurg PB, Carlsson AS, Whisson SC, Ortiz R (2021) Spray-induced gene silencing: an innovative strategy for plant trait improvement and disease control. Crop Breed Appl Biotechnol 21:e387921S11

Vinay Panwar VP, Jordan M, McCallum B, Bakkeren G (2018) Host-induced silencing of essential genes in Puccinia triticina through transgenic expression of RNAi sequences reduces severity of leaf rust infection in wheat. Plant Biotechnol J 16(5):1013–1023

Voloudakis AE, Kaldis A, Patil BL (2022) RNA-based vaccination of plants for control of viruses. Annu Rev Virol 9(1):521–548

Wang M, Weiberg A, Lin FM, Thomma BP, Huang HD, Jin H (2016a) Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nat Plants 2(10):1–10

Wang K, Peng Y, Pu J, Fu W, Wang J, Han Z (2016b) Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo. Insect Biochem Mol Biol 77:1–9

Wang Y, Guo Y, Guo S, Qi L, Li B, Jiang L et al (2024) RNA interference-based exogenous double-stranded RNAs confer resistance to Rhizoctonia solani AG-3 on Nicotiana tabacum. Pest Manag Sci 80(4):2170–2178

Werner BT, Gaffar FY, Schuemann J, Biedenkopf D, Koch AM (2020) RNA-spray-mediated silencing of Fusarium graminearum AGO and DCL genes improve barley disease resistance. Front Plant Sci 11:476

White FF, Potnis N, Jones JB, Koebnik R (2009) The type III effectors of Xanthomonas. Mol Plant Pathol 10(6):749–766

Worrall EA, Hamid A, Mody KT, Mitter N, Pappu HR (2018) Nanotechnology for plant disease management. Agronomy 8(12):285

Worrall EA, Bravo-Cazar A, Nilon AT, Fletcher SJ, Robinson KE, Carr JP et al (2019) Exogenous application of RNAi-inducing double-stranded RNA inhibits aphid-mediated transmission of a plant virus. Front Plant Sci 10:265

Xiong AS, Yao QH, Peng RH, Li X, Han PL, Fan HQ (2005) Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Rep 23:639–646

Yang XJ, Seto E (2011) The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol 9(3):206–218

Yoshioka M, Adachi A, Sato Y, Doke N, Kondo T, Yoshioka H (2019) RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre-and post-invasive resistance to potato blight pathogens. Mol Plant Pathol 20(7):907–922

Zhang J, Khan SA, Heckel DG, Bock R (2017) Next-generation insect-resistant plants: RNAi-mediated crop protection. Trends Biotechnol 35(9):871–882

Zhang T, Xu J, Chen J, Wang Z, Wang X, Zhong J (2021) Protein nanoparticles for pickering emulsions: a comprehensive review on their shapes, preparation methods, and modification methods. Trends Food Sci Technol 113:26–41

Zhu J, Dong YC, Li P, Niu CY (2016) The effect of silencing 20E biosynthesis relative genes by feeding bacterially expressed dsRNA on the larval development of Chilo suppressalis. Sci Rep 6(1):28697

Ziebell H, MacDiarmid R (2017) Prospects for engineering and improvement of cross-protective virus strains. Curr Opin Virol 26:8–14

Zotti M, Dos Santos EA, Cagliari D, Christiaens O, Taning CNT, Smagghe G (2018) RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Manag Sci 74(6):1239–1250

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Srimahesvari, D.S., Harish, S., Karthikeyan, G. et al. Advancements in dsRNA-based approaches: a comprehensive review on potent strategies for plant disease management. J. Plant Biochem. Biotechnol. (2024). https://doi.org/10.1007/s13562-024-00922-z

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DOI : https://doi.org/10.1007/s13562-024-00922-z

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