Applied Plant Virology: Advances, Detection, and Antiviral Strategies

Applied Plant Virology: Advances, Detection, and Antiviral Strategies provides an overview on recent developments and applications in the field of plant virology. The book begins with an introduction to important advances in plant virology, but then covers topics including techniques for assay detection and the diagnosis of plant viruses, the purification, isolation and characterization of plant viruses, the architecture of plant viruses, the replication of plant viruses, the physiology of virus-infected hosts, vectors of plant viruses, and the nomenclature and classification of plants. The book also discusses defense strategies by utilizing antiviral agents and management strategies of virus and viroid diseases.

With contributions from an international collection of experts, this book presents a practical resource for plant virologists, plant pathologists, horticulturalists, agronomists, biotechnologists, academics and researchers interested in up-to-date technologies and information that advance the field of plant virology.

• Covers the detection, control and management of plant viruses
• Discusses antiviral strategies, along with mechanisms of systemic induced resistance to enhance the defense of plants against viruses
• Provides contributory chapters from expert plant virologists from different parts of the world

• Language: English
• Publisher: Academic Press
• Release date: May 14, 2020
• ISBN: 9780128220535

Book preview

form.

Part 1

Important landmarks in the history of virology

Outline

Chapter 1 Major advances in the history of plant virology

Chapter 1

Major advances in the history of plant virology

Ahmed Hadidi¹, ², Panayota E. Kyriakopoulou³, ⁴ and Marina Barba⁵, ⁶,    ¹United States Department of Agriculture, Agricultural Research Service, Beltsville, MD, United State,    ²Emeritus Lead Scientist,    ³Department of Crop Science, Agricultural University of Athens, Athens, Greece,    ⁴Emeritus Professor,    ⁵CREA-Research Center for Plant Protection and Certification, Rome, Italy,    ⁶Emeritus Center Director

Abstract

The chapter deals with the history of the discovery of viruses in the late 19 century as well as the progress made over the last 120 years in advancing the science of plant virology. These include but are not limited to biology of virus-or viroid-infected plants, vector and non-vector transmission, the rise of molecular and biophysical virology, replication of RNA and DNA viruses as well as viroids, and many methods used in plant virology. These methods include serology, electron microscopy, confocal microscopy, analytical and preparative ultracentrifugation, density gradient ultracentrifugation, gel electrophoresis, hybridization. polymerase chain reaction, microarrays, genetic engineering, first and next-generation nucleotide sequencing, CRISPR-Cas system editing and several others. Finally, control of viruses and viroids by exclusion was also discussed.

Keywords

Virus discovery; tobacco mosaic virus; plant viruses; viroids; transmission; replication; molecular virology; methods in plant virology; control by exclusion

Chapter Outline

Outline

1.1 Introduction 4

1.2 Introduction of tobacco plants to Europe from the Americas 4

1.3 A tobacco disease in Europe that led to the beginning of virology 4

1.4 Discovery of plant DNA viruses, satellites, and viroids in the 20th century 5

1.4.1 DNA virus discovery 5

1.4.2 Viral satellites discovery 5

1.4.3 Viroid discovery 5

1.5 Virus-infected plant biology, the early years (1903–52) 5

1.6 Virus transmission 6

1.6.1 Nonvector transmission 6

1.6.2 Vector transmission 6

1.6.3 Viral protein involvement in aphid or nematode vector transmission 6

1.6.4 Propagative transmission 6

1.6.5 Transmission involving helper viruses 6

1.6.6 Circulative nonpropagative transmission 6

1.7 The beginning and rise of molecular virology with tobacco mosaic virus as a model system (1935–60) 8

1.8 The development of biophysical virology with tobacco mosaic virus as a model system (1937–89) 9

1.9 Replication 9

1.9.1 Replication of RNA viruses 9

1.9.2 Replication of DNA viruses 10

1.9.3 Viroid replication 10

1.10 Methods 10

1.10.1 Serology 10

1.10.2 Electron microscopy 10

1.10.3 Confocal microscopy 11

1.10.4 Analytical and preparative ultracentrifugation 11

1.10.5 Density gradient ultracentrifugation 11

1.10.6 Gel electrophoresis 11

1.10.7 Protoplast systems 12

1.10.8 A model plant susceptible to many viruses 12

1.10.9 Chemotherapy 12

1.10.10 Hybridization 12

1.10.11 Polymerase chain reaction 12

1.10.12 Microarrays 13

1.10.13 Genetic engineering 13

1.10.14 First-generation RNA sequencing 13

1.10.15 First-generation DNA sequencing 13

1.10.16 Next-generation sequencing 14

1.10.17 Next-generation sequencing of ancient viruses 14

1.11 Resistance to virus infection 14

1.11.1 Pathogen-derived resistance in transgenic plants 14

1.11.2 RNA silencing 14

1.11.3 Genome editing 15

1.11.4 CRISPR-Cas system editing confers resistance to plant viruses 15

1.12 Control by exclusion 16

References 17

Dedication: This chapter is dedicated to past and present scientists world-wide who contributed to the field of plant virology.

1.1 Introduction

The discovery of viruses in the late 19th century depended to a great extent on observations and discoveries made during the two millennia previous to that time. In ancient Greece, Theophrastus (c.370–287 BCE), considered to be the father of botany, in his Historia Plantarum (History of/Treatise on Plants), was the first to describe rust on grain crops (Gaza, 1483a); later Strabo (c.64 BCE to c.CE 24), in his Geographica wrote about the devastating effect of infection with rust (Gaza, 1483b). The Romans worshipped the rust god Robigus and sacrificed red dogs and cattle in order to mitigate the harmful effect of rust diseases on their grain crops (Large, 1940).

The first microscope was invented by Anton von Leeuwenhoek in 1683. In 1728, Henri-Louis Duhamel du Monceau demonstrated that a plant disease could be passed from plant to plant and that a contagious fungus was responsible for a saffron crocus disease (Duhamel de Monceau, 1728). In 1743, Needham discovered plant parasitic nematodes in wheat seeds in England.

The first detailed analyses of plant pathogens were described by Henrich Anton De Bary (1831–88), a German botanist, recognized as the father of modern plant pathology and the founder of modern mycology. In 1861, he proved that a fungus, Phytophthora infestans, was the causal agent of late blight disease of potato, and in 1863, he published a book identifying fungi as the cause, not the consequence, of a variety of plant diseases (De Bary, 1863). De Bary was credited for replacing the theory of spontaneously generated plant diseases with the germ theory of disease. In 1878, Thomas Jonathan Burrill (1839–1916), an American botanist/plant pathologist, was the first to discover that a bacterium, Erwinia amylovora, was associated with the fire blight disease in apples and pears (Burrill, 1878).

The microscope was crucial in the above discoveries and demonstrations that nematodes, fungi, and bacteria cause plant diseases. However, the discovery of viruses went through different stages without the help of the microscope, and the first observed virus disease was on tobacco plants that were introduced to Europe in the 16th century from the New World.

1.2 Introduction of tobacco plants to Europe from the Americas

Christopher Columbus, born Cristoforo Colombo (1451–1506), a famed Italian explorer and navigator, discovered the New World of the Americas in 1492 on an expedition sponsored by King Ferdinand of Spain. In the New World, Columbus observed that Native Americans smoked in pipes the leaves of a plant species originated in the Americas (tobacco) for medicinal purposes. For this reason, as well as for its being a successful cash crop in colonial America, the Spanish introduced tobacco to Europe about 1518. In 1571, it was claimed that tobacco could cure 36 health problems. In 1595, in addition to the medicinal benefits of tobacco, the health-giving properties of pipe smoking were emphasized (Hadidi and Barba, 2012).

1.3 A tobacco disease in Europe that led to the beginning of virology

When German chemist Adolf Mayer (1843–1942) was the director of the Agricultural Experiment Station in Wageningen, the Netherlands, he was asked by Dutch farmers to study a peculiar disease affecting tobacco plants; the disease caused tobacco to be extremely bitter to the taste (Harrison, 1952). He published his first paper on the disease in Dutch in 1882 and named it mozaïekziekte (mosaic disease), because of the variegation in the tobacco leaves, with light green and dark green patches (Mayer, 1882). Mayer casually remarked in his 1882 paper that perhaps a soluble, enzyme-like infectious principle was involved; however, he advocated the idea that a pathogenic bacterium was most likely the causal agent of the disease. In his subsequent publication in German 4 years later (Mayer, 1886), he demonstrated that the disease can be transmitted by using the sap from the affected tobacco plants as the inoculum to infect healthy plants; thus, he was the first to cause transmission of the disease experimentally. However, although he was able to transmit the contagious agent by mechanical means, he could not demonstrate the presence of a fungus or bacterium in diseased plants. Mayer did not carry out filtration experiments with sap from mosaic-diseased plants. It was Dmitri Ivanovski (1864–1920), a Russian botanist, who in 1892 was the first to show that extracts from mosaic-diseased tobacco plants could transmit disease to other plants after passage through ceramic filters that retain bacteria (Ivanovski, 1892). Charles Chamberland (1884), an associate of Louis Pasteur, developed the porcelain bacterial filter used by Ivanovski. Ivanovski is considered to be the discoverer of the viruses.

In 1898, Martinus Beijerinck, a Dutch microbiologist and botanist, confirmed and extended Ivanovski’s results. He concluded that tobacco mosaic disease is caused by an infectious agent that is smaller than a bacterium and replicates in living plants; he named it virus and referred to it as contagium vivum fluidum (soluble living germ or contagious living fluid) (Beijerinck, 1898). Thus, tobacco mosaic disease is caused by tobacco mosaic virus (TMV). It is important to note that Beijerinck made a clear distinction between the filterable agent and a contagium vivum fixum, as coined by Pasteur for pathogenic microorganisms. This is generally recognized as the beginning of virology.

In 1898 in Germany, Freidrich Loeffler and Paul Frosch (former students of Koch) reported that a similar agent (virus) was the responsible contagious agent of the foot-and-mouth disease of cattle. The agent was passed through a Kieselguhr filter (which is comparable to a porcelain filter), and the filtrate was free of microorganisms (Loeffler and Frosch, 1898). This study was the first report that an animal pathogen was filterable and, unlike bacteria, did not grow on artificial media. The first filterable agent to be discovered in humans was yellow fever virus in 1901, and it was found to be transmitted by mosquitoes (Levine and Enquist, 2007). Thus, viruses have been shown to cause diseases in plants, animals, and humans.

1.4 Discovery of plant DNA viruses, satellites, and viroids in the 20th century

1.4.1 DNA virus discovery

In 1968, Robert J. Shepherd and colleagues reported the first plant virus with a DNA genome, cauliflower mosaic virus (CaMV) (Shepherd et al., 1968).

1.4.2 Viral satellites discovery

Basil Kassanis coined the term satellite for very small virus particles he found in some cultures of tobacco necrosis virus (TNV). He demonstrated that these spherical 17-nm viral satellite particles were unable to replicate in the absence of the larger 30-nm TNV particles (Kassanis and Nixon, 1961). Similarly, in 1971, Irving R. Schneider found nonessential but biologically active satellite RNA molecules in preparations of various other viruses (Schneider, 1971). In 1972, a devastating epidemic disease swept through tomato fields of the French Alsace province destroying the crop. The causal agent was not revealed until 1977, when Jacobus M. Kaper (Kaper and Waterworth, 1977) clearly demonstrated that RNA 5 of cucumber mosaic virus (CMV) was required to cause the Alsace disease. Because RNA 5 alone cannot cause the disease, but needed other CMV RNAs for replication, this CMV RNA was recognized as the first RNA satellite to cause a plant disease.

1.4.3 Viroid discovery

Theodor O. Diener demonstrated that the potato spindle tuber agent was a free RNA without a protein coat and weighed only 25,000–110,000 Da (Diener, 1971), much smaller than a viral genome, and named it viroid a year later. Independently, Joseph S. Semancik discovered that the causal agent of citrus exocortis disease has properties similar to those of the causal agent of potato spindle tuber disease (Semancik and Weathers, 1972). Diener remarked that the term viroid had earlier been put forward by Altenburg (1946) to describe possible ...symbionts akin the viruses... in animal cells.

1.5 Virus-infected plant biology, the early years (1903–52)

During the first decades of the 20th century, virus disease external systemic symptoms, including mosaic and leaf curl were described in plant crops, including potatoes, tobacco, legumes, cucurbits, sugar beets, and others. These symptoms become systemic, spreading to whole plants and to their vegetative progenies. In addition, internal symptoms, such as crystalline and amorphous inclusion bodies inside TMV-infected leaves, were described (Ivanovski, 1903).

Necrotic local lesion formation on tobacco leaves mechanically inoculated with TMV was discovered by Francis Holmes (1929), who showed that it could be the basis of an improved infectivity assay because the number of lesions developed depends on the virus titer in the inoculum. He also reported that local lesion response to virus infection was an effective form of resistance controlled by a single dominant gene (Holmes, 1934).

McKinney (1929) demonstrated that TMV mutates, as he found a variant or mutant of TMV that could be cultured from yellow spots that appeared in some systemically infected tobacco leaves. In addition, in the same paper, he reported the phenomenon of cross-protection, in which plants infected with one strain of a virus were resistant to subsequent infection by a second more virulent strain of the same virus, however, these plants remained susceptible to infection by other viruses.

Samuel (1934) and Bennett (1937) studied the movement of TMV and curly top virus, respectively, each within its host plants. It was found that cell-to-cell movement was through plasmodesmata, whereas long-distance movement was through the phloem sieve tubes, first to young roots and shoots then to mature tissues.

Thermotherapy (heat therapy) (Kassanis, 1950) and in vitro apical meristem culture (Morel and Martin, 1952) have been successfully used for eliminating virus infection from mother clone stocks of vegetatively propagated plant species, and increasing numbers of virus-resistant crop cultivars were bred. The standard method for virus elimination in many vegetatively propagated crops is thermotherapy combined with meristem culture.

1.6 Virus transmission

1.6.1 Nonvector transmission

Studies of transmission of plant viruses are important in understanding disease epidemiology, which is a prerequisite for developing effective virus-control measures. Grafting was known among Dutch growers in the 17th century to transmit the broken-tulip condition, now known to be a virus infection. Also, grafting and budding are centuries-old methods in many countries for disseminating vegetatively propagated crops, and many viruses in these crops are transmitted by grafting. Natural root and shoot grafting also plays a role in virus transmission. Mechanical transmission of TMV was first reported using sap from infected plants in capillary tubes (Mayer, 1886). Subsequently, glass rods and other tools were used for transmission of viruses using sap. The efficiency of mechanical transmission increased by dusting plant leaves with fine carborundum prior to inoculation (Rawlins and Tompkins, 1936). In addition to virus-infected sap, mechanical transmission may occur by other means, such as through the use of farming tools and agricultural practices.

1.6.2 Vector transmission

It was revealed as early as 1901 and 1919 that insects and plants, respectively, are vectors for the transmission of plant viruses and that they play a crucial role in plant virus epidemiology. Subsequently, they were also discovered to transmit viroids. Table 1.1 shows landmarks in vector transmission of plant viruses and viroids.

Table 1.1

It is worth noting that potato spindle tuber viroid (PSTVd) is transmitted in a nonpersistent manner by the aphid Macrosiphum euphorbiae (De Bokx and Pirone, 1981) and in a persistent manner by the aphid Myzus persicae when it is encapsidated in a potato leaf roll virus particle (Salazar et al., 1995; Querci et al., 1997).

In addition to the vectors presented in Table 1.1, it has been reported that some viruses that belong to tombus-, potex- and tobamovirus groups as well as other viruses of unknown groups are waterborne, as they were isolated from rivers and lakes in Germany (Koenig and Lesemann, 1985).

1.6.3 Viral protein involvement in aphid or nematode vector transmission

Different viruses with different lengths of persistence in aphid vectors may rely for transmission on the specificity of one or two nonstructural viral proteins, as well as on the specificity of their coat protein (CP). Potyviruses are transmitted with the help of a virus-coded nonstructural protein known as helper component protein (HC-Pro), which binds the virus particles to aphid mouthparts (Thornbury et al., 1985; Berger and Pirone, 1986; Ammar et al., 1994).

Caulimoviruses persist in aphid vectors for several hours and also use an attachment mechanism using the association of virus particles with aphid mouthparts. An HC-Pro (Woolston et al., 1987) called P2 binds to aphid stylets and to a second viral nonstructural protein termed P3 (Leh et al., 1999). Binding of the P3-virus particle complex to P2 presumably occurs in the aphid, after P2 attaches to the aphid mouthparts (Drucker et al., 2002). For transmission to occur, this binding must occur before the P3-virus complex interacts with P2. Some styletborne viruses such as cucumoviruses, however, do not need an HC-Pro for nonpersistent transmission by aphids. The viral CP is the only protein needed (Chen and Francki, 1990).

An HC-Pro, the 2b protein, was also found to be needed for the persistent noncirculative transmission of tobraviruses by trichodorid nematodes (MacFarlane et al., 1996). The 2b protein binds in cells to the viral C-terminal region of the CP; it is vital for the interaction and for transmission (Vellios et al., 2002).

1.6.4 Propagative transmission

Ullman et al. (1993) and Wijkamp et al. (1993) showed clear evidence that tospoviruses (family Bunyaviridae) replicate in their vectors, thrips. Thus, tospoviruses are similar to vertebrate-infecting bunyaviruses in that they are able to infect their arthropod vectors.

1.6.5 Transmission involving helper viruses

Two umbraviruses (Falk et al., 1979; Waterhouse and Murant, 1983), a variety of satellite RNA (Rasochova et al., 1997), viroid (Querci et al., 1997) and luteovirus-associated RNA (Falk and Duffus, 1984), rely on propagative transmission, as they can be packaged and become aphid-transmissible.

1.6.6 Circulative nonpropagative transmission

Caulimoviruses, tobraviruses, cucumoviruses, and luteoviruses have been known to circulate through the body of their aphid vectors but do not replicate during transmission. In the case of barley yellow dwarf virus, the aphid vector salivary gland surface recognizes the virus CP readthrough domain, enabling virus particles to enter the salivary gland cells (Gildow and Gray, 1993). This readthrough domain interacts in aphid hemolymph with the protein symbionin, and this interaction seems important in the prevention of inactivation of virus particles. That may explain the long persistence of luteoviruses without replication in aphid vectors (van den Heuvel et al., 1994, 1997).

Another form of persistent circulative transmission is that of beet necrotic yellow vein virus (BNYVV), a benyvirus that is transmitted through the roots by Polymyxa betae, a plasmodiophorid vector. Plasmodiophorids are now classified as protists. The virus can be retained in resting spores of P. betae, apparently without replicating. BNYVV RNA 3 encodes a protein that enhances invasion of root tissues and causes a root proliferation known as rhizomania (Tamada et al., 1999) and RNA 4 encodes a protein that is needed for transmission by the vector (Tamada and Abe, 1989). RNA 3 and RNA 4, separately or in combination, greatly increase transmission frequency. BNYVV CP, encoded by RNA 2, has a readthrough domain. The C-terminal region of this domain is needed for transmission by P. betae (Tamada and Kusume, 1991). The importance of this readthrough domain for transmission is very similar to that in the luteovirus system.

1.7 The beginning and rise of molecular virology with tobacco mosaic virus as a model system (1935–60)

TMV was the first virus to be thoroughly analyzed, beginning a generation of scientists’ work describing the molecular structures and replication of other viruses. Wendell M. Stanley, an American biochemist, was the first to purify and crystallize a virus that infects plants (TMV) or humans (influenza virus). He purified, crystallized, and described the molecular structure of TMV in 1935 (Stanley, 1935), showing that it has properties of both living and nonliving matter (Stanley, 1935, 1936a,b). He obtained a virus suspension preparation that he described as a crystalline protein possessing the properties of TMV. (Stanley, 1937). On dissolution of the crystals, the suspension preparation obtained was infectious, also after repeated recrystallizations. He won the Nobel Prize for Chemistry in 1946, sharing the honor with John H. Northrop and James B. Summer.

Stanley’s achievements with TMV were soon reproduced in England by plant pathologist Frederick Bawden and biochemist Norman Pirie in 1936 (Bawden et al., 1936), who also reported the presence of 0.5% phosphorus and 2.5% carbohydrate in TMV crystals, which suggested the presence of nucleic acid of the ribose type.

Arthur C. Knight (1947), an American biochemist, showed that the protein in particles of a yellow TMV variant discovered by McKinney (1929) had an altered amino acid composition; thus, viruses mutated and the mutants could be subcultured and distinguished chemically.

From the early 1950s to 1956, Heinz Fraenkel-Conrat, an American biochemist, was the first to demonstrate that replication of a virus (TMV) is controlled by genetic information within its RNA core. He proved conclusively that TMV RNA genetic material was the infectious agent and that the protein coat had no infective properties. He was also the first scientist to show that virus molecules, still retaining viral life, could thus be reconstituted from its separated protein and RNA. Fraenkel-Conrat and Williams (1955) described the reassembly of TMV particles from their isolated protein and RNA components and showed that the reassembled particles were infective. Fraenkel-Conrat also discovered that he could disassemble and reassemble TMV by adding and removing detergents (Fraenkel-Conrat, 1956). In subsequent studies, he showed that TMV RNA, once inside the cell, took over the host cell’s own genetic material (DNA) and began replicating, making not only more infective RNA but compatible protein coatings as well. This study provided definitive proof that RNA can act like DNA as the genetic blueprint for cell replication. In 1957, Fraenkel-Conrat and Singer published the famous mixed reconstitution experiment, taken as the final proof that nucleic acids encode protein structure, and not the opposite (Fraenkel-Conrat and Singer, 1957). In 1960, Fraenkel-Conrat and colleagues announced the complete amino acid sequence of TMV protein, consisting of 158 amino acid residues, making it the biggest protein of known structure at the time (Tsugita et al., 1960). Fraenkel-Conrat’s accomplishments were recognized with numerous awards and honors, including the Albert Lasker Award, the first California Scientist of the Year Award in 1958, and many others, including The Pope’s Pontifical Medal.

Gierer and Schramm (1956), in Germany, also proved that phenol-purified TMV RNA was infective. Subsequently, Gierer and Mundry (1958) produced TMV RNA mutants by treating the purified RNA with nitrous acid to deaminate its nucleotide bases; this resulted in the amino acid replacements of the mutant CPs. TMV mutants, especially those developed by Tsugita (1962) and Wittmann (1962), helped to accurately crack the genetic code.

1.8 The development of biophysical virology with tobacco mosaic virus as a model system (1937–89)

Bernal and Fankuchen (1937) used X-ray diffraction to study TMV suspension. They showed that TMV suspension consisted of particles and that each particle had a width of approximately 15 nm and a length of at least 10 times the width. They also deduced that each viral particle consisted of identical, regularly arranged subunits.

Watson (1954), using X-ray diffraction, showed that TMV particles are helical. Franklin (1955, 1956) and Caspar (1956) confirmed Watson’s finding and provided evidence that the helix was hollow rather than solid and that TMV RNA was embedded in the protein helix. Each turn of the helix has 16⅓ protein subunits (Franklin and Holmes, 1958). The above studies led to the discovery of the helical structure of TMV particles, the first for a virus.

Fraenkel-Conrat (1956) demonstrated that TMV particles were broken down with detergents and then reconstituted by removing the detergents at pH 7 and ionic strength 0.5. In these experiments, he noted the appearance of disks with central holes and that each disk was arranged in two layers. Furthermore, evidence showed that TMV CP disks comprising two cylindrical layers of 17 subunits might serve as an important intermediate for the assembly of virus helixes (Lauffer et al., 1958; Caspar, 1960). Butler and Klug (1978) showed that TMV CP disks are essential for self-assembly, which led them to present a model of assembly involving first nucleation and then elongation. In this manner, the helical TMV particle is assembled, consisting of one piece of single-stranded RNA lying within a groove by the helical array of CP subunits. Over time, the protein subunits realign and anneal into an uninterrupted helical rod (Butler, 1984). Atomic resolution of intact TMV provided new structural details of both the RNA and CP subunits and a 2.9-Å map of TMV was obtained (Namba et al., 1989).

1.9 Replication

1.9.1 Replication of RNA viruses

The first RNA-dependent RNA polymerase (RdRp) to be characterized was that of the bacteriophage MS2 in 1963; it was termed replicase (Haruna et al., 1963). Ten years later, brome mosaic virus (BMV), the first RdRp replicase of a virus that infects eukaryotes, a plant virus, was identified and characterized from virus-infected barley seedlings by Ahmed Hadidi (Hadidi and Fraenkel-Conrat, 1973; Hadidi et al., 1973; Hariharasubramanian et al., 1973; Hadidi, 1974). A protein of 34,500 Da was detected as a component of BMV RdRp using the double-labeling approach and sodium dodecyl sulfate (SDS)-gel electrophoresis analysis (Hariharasubramanian et al., 1973). The pioneering work on the BMV RdRp has been confirmed and extended to different systems; it has also been extended to other plant viruses with single-stranded RNA (ssRNA) genomes. Most plant viruses have ssRNA genomes of positive sense, which may include members of several economically important families such as All virus or viroid family names should be in italic according to the Code of Virus Classification and Nomenclature established by the International Committee on Virus Taxonomy (ICTV). Please convert all family names throughout the text into italic. Examples: Potyviridae should be Potyviridae; Betaflexiviridae should be Betaflexiviridae Potyviridae, Bromoviridae, Virgaviridae, Tombusviridae, Secoviridae, Luteoviridae, Tymoviridae, Closteroviridae, Alphaflexiviridae, and Betaflexiviridae (Sastry, 2013).

Using DNA templates to characterize the requirements for BMV RdRp template recognition, Sivakumaran and Kao (1999) showed that initiation from the 3′ end of a template requires one nucleotide 3′ of the initiation nucleotide. The addition of a single nontemplated nucleotide at the 3′ end of minus-strand BMV three RNAs led to initiation of genomic plus-strand RNAs in vitro. Collmer and Kaper (1985) were the first to show that double-stranded RNAs (dsRNAs) of CMV and its satellite contain an unpaired terminal guanosine, which, together with elements further downstream, is essential for RdRp recognition.

Some plant viruses have ssRNA of negative sense, such as members of the families Rhabdoviridae, Bunyaviridae, and Ophioviridae. The basic mechanism of replication of a negative-sense RNA genome is very similar to that of the positive-sense RNA except that the viral RdRp uses the negative strand as a template for synthesizing a complementary positive strand and then a new negative strand is synthesized. The positive-sense RNA, which acts as a viral mRNA, is translated into proteins for the production of new virions (Ortin and Martin-Benito, 2015).

1.9.2 Replication of DNA viruses

Replication of the dsDNA genome of CaMV involves reverse transcription (RT), and the full-length transcript is an intermediate in this process (Guilley et al., 1983; Hull and Covey, 1983; Pfeiffer and Hohn, 1983). CaMV open reading frame (ORF) V codes for reverse transcriptase (Toh et al., 1983; Takatsuji et al., 1986). All dsDNA plant viruses, including badnaviruses have a replication strategy similar to that of CaMV, involving RT.

Replication of the DNA genomes of geminiviruses is by a rolling-circle mechanism (Stenger et al., 1991). The only virus-coded protein that is required is a replication initiator protein known as Rep (Laufs et al., 1995).

1.9.3 Viroid replication

Being nonprotein-coding RNAs, viroids replicate by manipulating preexisting cellular RNA polymerases and processing enzymes in host nuclei (members of the family Pospiviroidae) or chloroplasts (members of the family Avsunviroidae) (Hadidi et al., 2003, 2017). The identification of multimeric minus (−) viroid RNAs (complementary to the predominant infectious [+] strand) in PSTVd-infected tomatoes (Branch et al., 1981) and of double-stranded dsDNA PSTVd complexes containing unit- and longer-than-unit-length (+) and (−) strands (Hadidi et al., 1982), as well as multimeric (+) viroid RNAs in avocado sunblotch viroid-infected avocado (Bruening et al., 1982), led to the proposal that one or both of these longer-than-unit-length strands resulted from the reiterative transcription of circular templates through a rolling-circle mechanism (Branch and Robertson, 1984) with only RNA intermediates (Grill and Semancik, 1978; Hadidi et al., 1981, 1982). Viroid longer-than-unit -strands are catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase. Cleavage to unit- length in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in viroid strands, while in the family Pospiviroidae only the oligomeric (+) RNAs provide the proper conformation to be cleaved by an RNase of class III and iii. Circularization of the unit- length is catalyzed by DNA ligase I (family Pospiviroidae) or by a chloroplastic isoform of transfer RNA (tRNA) ligase (family Avsunviroidae) (Flores et al., 2017). These enzymes (and ribozymes) are most likely assisted by other host proteins.

1.10 Methods

1.10.1 Serology

In the late 1920s and early 1930s, investigators such as Helen Purdy (Purdy, 1928) and Gratia (1933a,b) established that plant viruses acted as antigens. Thus, a new way to detect virus infection was found. Histochemical methods were developed for detecting pathogens using fluorescent antibodies (Coons et al., 1942). Gel-diffusion tests were developed for detection of a human bacterial pathogen (Ouchterlony, 1948) and then were used for virus study and detection (Ouchterlony, 1949, 1968). In addition, Grabar and Williams developed the immunoelectrophoresis technique (Grabar and Williams, 1953). That led to the development of more serological assay methods to detect viruses in infected plants (Van Slogteren and Van Slogteren, 1957). Ball and Brakke (1968) were the first to describe the leaf-dip technique for virus detection using electron microscopy (EM). This technique then was improved by others and renamed serologically specific EM, immunosorbent EM, or immune EM (Cambra et al., 2011). The radioimmunology was developed by Yalow and Berson (1960). Monoclonal antibodies—hybrid myelomas—a revolution in serology and immunogenetics were developed by Köhler and Milstein (1975) and were subsequently used in research on plant virology. The enzyme-linked immunosorbent assay (ELISA) was first used for detecting two plant viruses, arabis mosaic virus and plum pox virus (Voller et al., 1976). A year later, the ELISA microplate method was developed for detecting plant viruses (Clark and Adams, 1977).

1.10.2 Electron microscopy

Debates about possible origins of viruses stimulated biophysicists to characterize the virus particles in more detail. TMV was the first virus to be viewed using EM (Kausche et al., 1939). EM examination showed rod-shaped particles, approximately 15 nm wide and 300 nm long. Subsequently, the morphology of virus particles of a large number of viruses were characterized by EM examination of their preparations making use of improved purification techniques as well as improvement in the resolution of EM and the development of EM techniques. The first of these techniques was shadow casting (Muller, 1942; Williams and Wyckoff, 1945) which was followed by others such as the negative staining technique (Brenner and Horne, 1959). In addition, the location of virus particles in infected tissues and cells could be studied by EM in ultrathin sections (Bretschneider, 1950; Hall, 1964). The freeze-etch and freeze-fracture EM techniques were developed by Russell L. Steere for revealing three-dimensional ultrastructure information on viruses and cells not readily obtained from thin sections (Steere, 1957, 1989; Steere and Erbe, 1979; Steere et al., 1980).

There are three main types of EM—transmission, scanning, and emission (Hari et al., 2011). Transmission electron microscopy (TEM) is the main choice in studying viruses and virus–plant-cell interactions. TEM is also used for immunoelectron microscopy. In 2019, EM in combination with bioinformatics allowed the transition from 2D imaging to 3D remodeling, which creates detailed structural and functional analyses of viruses (Richert-Pöggeler et al., 2019).

1.10.3 Confocal microscopy

The principle of confocal microscopy was described and patented by Marvin Minsky in 1957 (1961 US patent number 3,013,467). However, it took more than three decades to develop and use the confocal microscope for the examination of biological samples, including virus-infected tissues. One of the major advantages of confocal microscopy is its ability to obtain point-by-point in-focus images of several layers of cells in a thick section sample through serial optical sections, and a 3D image can be constructed by means of a computer attached to the microscope using image analysis software. The most common use of confocal microscopy is through the use of one or more fluorescent probes, although unstained samples can also be used (see Hari et al., 2011, for references published in the 1990s and in 2000s).

Laser scanning confocal microscopy is used to obtain digital images of samples labeled with as many as three different fluorochromes and to identify colocalization when two different fluorochromes label a single object. The method has been used to study virus–plant-cell interactions, especially in the localization of virus-movement proteins (Boyko et al., 2002; Haupt et al., 2005; Liu et al., 2005; Wei et al., 2006; Ghazala et al., 2008).

1.10.4 Analytical and preparative ultracentrifugation

The analytical ultracentrifuge was invented by Theodor Svedberg in 1925; he won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge. One of the major advantages of the analytical ultracentrifuge is that it can provide a physical criterion identity for viruses, the sedimentation coefficient in Svedberg units. It is also a valuable method for studying the purity of virus preparations and the effects of various treatments on the physical state of the virus. It is also valuable for assaying the amounts of virus in crude samples.

The vacuum preparative ultracentrifuge was invented by Edward G. Pickles. The centrifuge allowed a reduction in friction generated at high speed and allowed the maintenance constant temperature across the sample. In 1946, Pickles cofounded Spinco (Specialized Instruments Corporation) and marketed a vacuum ultracentrifuge. In 1947, Spinco was the first company to commercially manufacture ultracentrifuges. In 1949, Spinco introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of 40,000 rpm. In 1954, Beckman Instruments (now Beckman Coulter) purchased the company, forming the basis of its Spinco centrifuge division, which has developed both preparative and analytical centrifuges.

1.10.5 Density gradient ultracentrifugation

The application of ultracentrifugation, especially density gradient centrifugation developed by Myron K. Brakke, an American biochemist, has been a great asset in improving virus isolation and purification techniques (Brakke, 1951, 1960). This technique, which is important for virus characterization, is based on the fact that sedimentation of virus particles depends not only on their mass, morphology, and density, but also on the density of the medium. It has proven to be a highly versatile technique and is widely used in the field of virology and molecular biology. Density gradient centrifugation has largely replaced analytical ultracentrifugation in virus studies.

1.10.6 Gel electrophoresis

Gel electrophoresis methods have diversified significantly, and new methods and applications have been developed since the 1950s (Vesterberg, 1989).

When native proteins are dissolved in denaturing solvents, denatured proteins do not aggregate and become unambiguous and easy to analyze. SDS, which was introduced into biochemistry in 1938 to disrupt TMV, is one of the most useful solvents for denaturing proteins (Sreenivasaya and Pirie, 1938). SDS was used to develop SDS polyacrylamide gel electrophoresis (SDS-PAGE) for protein molecular weight analysis. SDS-PAGE was used with great success for a wide variety of proteins (Weber and Osborn, 1969). The gel electrophoresis method was improved to detect and identify proteins of bacteriophage T4 (Laemmli, 1970). A few years later, SDS-PAGE in tubes was used in plant virology for the analysis of TMV-induced proteins (Zaitlin and Hariharasubramanian, 1972) and identification and determination of the molecular weight of a component of brome mosaic virus RdRp (Hariharasubramanian et al., 1973) in infected plants using the double-labeling approach.

PAGE was used for viroid identification in infected plants; it was used to identify PSTVd in infected tomatoes (Diener, 1971).

The first Western blot was developed by Towbin et al. (1979). Rybicki and von Wechmar (1982) were the first to combine the fractionation of plant viral protein by gel electrophoresis and the sensitivity and specificity of a solid phase immunoassay to identify the virus CP by two independent properties, molecular weight and serological specificity.

1.10.7 Protoplast systems

Tobacco mesophyll protoplasts infected with TMV, developed by Takebe and Otsuki (1969), for the first time allowed studies of single-step virus growth curves.

1.10.8 A model plant susceptible to many viruses

Antonio Quacquarelli, an Italian plant virologist was the first to report that Nicotiana benthamiana Domin is susceptible to many viruses (Quacquarelli and Aveglis, 1975). Since then, it has been used extensively in plant virus research. Currently, it is the most widely used experimental host in plant virology.

1.10.9 Chemotherapy

Chemotherapy was developed to eliminate potato viruses by adding ribavirin (an antiviral compound) to the culture media and isolating the axillary buds (Griffiths et al., 1990). Ribavirin alone or in combination with thermotherapy (Kassanis, 1950) was effective, and virus-free plants were obtained from both treatments.

1.10.10 Hybridization

Gillespie and Spiegelman (1965) were the first to show that DNA immobilized on a membrane can bind a complementary RNA or DNA strand through specific hybridization. Subsequently, Southern and Mitchell (1971) described methods for applying DNA to a treated cellulose surface and DNA blotting hybridization (Southern, 1975). The later technique has been known since then as Southern blotting. Kafatos et al. (1979) described the determination of nucleic acid sequence homologies and relative concentration by dot blot hybridization. In 1977, RNA blotting (sometimes termed Northern blotting) was developed (Alwine et al., 1977). It is similar to Southern blotting except that RNA, rather than DNA, is analyzed. These methods illustrate the concept of utilization of probes detected by a signal, whether radioactive or fluorescent.

1.10.11 Polymerase chain reaction

During the winter of 1983–84, polymerase chain reaction (PCR), which uses a thermostable DNA polymerase, was developed by Kary Mullis (Mullis et al., 1986; Saiki et al., 1988), who received the Nobel Prize for Chemistry in 1993 for this discovery. The PCR is best described in the words of US patent number 4,683,202: The process comprises treating separate complementary strands of the (target) nucleic acid with a molar excess of two oligonucleotide primers and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence.

The automated DNA thermal cycler was introduced in 1989, and it had the ability to amplify in vitro specific DNA or complementary DNA (cDNA) sequences from trace amounts in a complex mixture of templates. It was possible at that time to amplify specific DNA or cDNA sequences from as short as 50 bp to over 10,000 bp in length, more than a million-fold in a few hours (10⁶- to 10⁹-fold amplifications in 3–4 hours or less). In the same year, this technology was used by Ahmed Hadidi to show for the first time that RT-PCR may be used for the sensitive detection of pome fruit viroids as well as viral satellite RNA and temperate fruit viruses (Hadidi and Yang, 1990). The detection of viroids by RT-PCR requires 1–100 pg of total nucleic acids from infected tissue and generally is 10- to 100-fold more sensitive than viroid detection by hybridization using complementary RNA (cRNA) probes and 2500-fold more sensitive than return gel electrophoresis analysis (Hadidi and Yang, 1990). PCR has been established since 1990 as one of the most substantial technical advances in molecular biology, particularly in plant pathogen identification, detection, and diagnosis. Its current applications are in the areas of disease diagnosis, detection of pathogens, detection of DNA in small samples, DNA comparison and mutation detection, high-efficiency cloning of genomic sequences and numerous other areas. PCR has thus impacted basic molecular, biological, and clinical research as well as forensics, evolutionary studies, genome projects, and plant pathology, including plant virology (Faggioli et al., 2017; Hadidi et al., 1995, 2011; Candresse et al., 1998; Hadidi and Candresse, 2001, 2003). Loop-mediated isothermal amplification of DNA (LAMP) was developed in 2000 (Notomi et al., 2000). Reverse transcription–LAMP (RT-LAMP) was successfully used for the detection of RNA viruses (Fukuta et al., 2005).

Viroids are the first plant pathogens for which RT-PCR was used (Hadidi and Yang, 1990). Their amplification methods have increased in number with the development and application of real-time RT-PCR (Boonham et al., 2004), RT-LAMP (Boubourakas et al., 2009; Fukuta et al., 2005) and the combination of techniques, such as RT-PCR–ELISA (Shamloul et al., 2002), multiplex RT-PCR (Ito et al., 2002; Levy et al., 1992), multiplex bead-based array (van Brunschot et al., 2014), and in situ RT-PCR (Boubourakas et al., 2011).

1.10.12 Microarrays

Schena et al. (1995) published the first paper describing microarrays for quantitative measuring of gene expression. Microarrays were introduced as a tool for measuring the levels of gene expression of multiple genes in a high-throughput, parallel mode, alongside techniques such as quantitative PCR, serial analysis of gene expression, and others (Hadidi et al., 2004). Base-pairing of complementary sequences by hybridization is the underlying principle of DNA microarray techniques. This specific binding of DNA allows a target DNA or RNA to hybridize to a specific cDNA probe on the array. Each probe is made of thousands of cDNAs or oligonucleotides, each specific for a gene, DNA sequence or RNA sequence of interest (Hadidi et al., 2004). Wang et al. (2002) developed a DNA microarray capable of simultaneously detecting about 140 human and animal viruses, including double- and single-stranded DNA viruses, retroviruses, as well as both positive- and negative-stranded RNA viruses. Microarrays were used for the first time for plant virus detection in 2003 (Hadidi et al., 2004), for virus genotyping in 2008 (Barba and Hadidi, 2011), and for viroid detection in 2012 (Tiberini and Barba, 2012). In addition to detection, microarray-based functional genomics has been widely used to monitor changes in host transcription during plant–virus or plant–viroid interaction (Owens et al., 2017).

Microarrays allow the simultaneous detection of hundreds of diverse viruses and many viroids (for review, see Barba and Hadidi, 2011; Zhu et al., 2017). Related pathogens may be distinguished by their unique pattern of hybridization. Moreover, by selecting microarray elements derived from highly conserved regions within pathogen families, genera, or groups, individual pathogens that are not explicitly represented on the DNA microarray are still detected, thus, this approach may be used for pathogen discovery. The DNA microarray method is versatile and greatly expands the spectrum of detectable viruses and viroids, in a single assay while simultaneously providing the capability to discriminate among the pathogens detected. This technology has been used in different aspects of plant virology research.

1.10.13 Genetic engineering

In 1972, Paul Berg, an American biochemist, developed DNA technology, which permits isolation of defined fragments of DNA (Jackson et al., 1972). Prior to this, the only accessible samples for sequencing were from phage or virus DNA. Also, his discovery led to the development of modern genetic engineering. The 1980 Nobel Prize in Chemistry was divided, with half awarded to Paul Berg for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA, and the other half jointly to Walter Gilbert and Frederick Sanger for their contributions concerning the determination of base sequences in nucleic acids.

1.10.14 First-generation RNA sequencing

In 1964, American biochemist Robert W. Holley (1922–93) was the first to sequence an RNA. He determined the complete sequence and structure of the 77 ribonucleotides of alanine transfer RNA (tRNA) the molecule that incorporates the amino acid alanine into protein (Holley et al., 1964, 1965). Holley’s pioneering work opened the door for others to determine the sequence of other RNAs as well as DNAs. The 1968 Nobel Prize in Physiology or Medicine was awarded jointly to Robert W. Holley, Har Gobind Khorana, and Marshall W. Nirenberg for their interpretation of the genetic code and its function in protein synthesis.

In 1976, Walter Fiers, a Belgium molecular biologist, was the first to establish the complete sequence of the 3569 nucleotides of bacteriophage MS2 RNA (Fiers et al., 1976).

1.10.15 First-generation DNA sequencing

In 1973, the first nucleotide sequence of 24 bp out of 27 bp of the lac operator DNA was published (Gilbert and Maxam, 1973). In 1977, Frederick Sanger, a British biochemist was the first to sequence the complete DNA genome of bacteriophage ΦX 174 (Sanger et al., 1977a). He also developed DNA sequencing with chain-terminating inhibitor (Sanger et al., 1977b). Also in 1977, Walter Gilbert, an American biochemist and physicist, developed DNA sequencing by chemical degradation (Maxam and Gilbert, 1977). Walter Gilbert and Frederick Sanger shared the 1980 Nobel Prize in Chemistry with Paul Berg, as mentioned above.

The classical first-generation Sanger shotgun sequencing (Sanger et al., 1977a,b) was first used for complete genome sequencing of a plant DNA virus, CaMV (Frank et al., 1980), an RNA virus, TMV (Goelet et al., 1982), and a viroid, PSTVd (Gross et al., 1978). TMV ORFs identified by sequencing (Goelet et al., 1982) confirmed previously obtained results, but in the cases of some other viruses, it took longer to clarify the coding potential of the virus sequencing. The ORFs define the potential capacity of a virus to produce proteins.

One of the most significant landmarks using Sanger sequencing was the discovery that the RNA of the potyvirus tobacco etch virus contains one very large ORF (Allison et al., 1986), which encodes a polyprotein that is processed to give the individual virus proteins. Subsequently, synthesis and processing of a polyprotein have been shown to be the mode of expression of the RNAs of several other groups of viruses, in addition to potyvirus RNAs.

1.10.16 Next-generation sequencing

Next-generation sequencing (NGS), which was developed at the beginning of the 21st century (for details, see Hadidi and Barba, 2012; Barba et al., 2014), is a powerful tool that has provided dramatic improvement in sequencing speed and depth together with very significant decline in costs as compared with traditional first-generation Sanger sequencing technologies; combined with recent developments in applicable bioinformatics these technologies have dramatically changed the field of plant virology. They were first used in plant virology in 2009 (Adams et al., 2009; Al Rwahnih et al., 2009; Di Serio et al., 2009; Donaire et al., 2009; Kreuze et al., 2009; Navarro et al., 2009) and have been used in the past several years for genome sequencing, ecology, discovery, evolution, population biology, epidemiology, transcriptomics, replication, mutation detection, identification, and others (Barba et al., 2014; Barba and Hadidi, 2015; Hadidi et al., 2016; Hadidi, 2019).

1.10.17 Next-generation sequencing of ancient viruses

Smith et al. (2014) were the first to use NGS technology to sequence the whole genome of an isolate of barley stripe mosaic virus (BSMV) from barley grains that were approximately 750 years old. The sequence obtained did not fit well with the phylogenetic reconstruction of the evolutionary time line for BSMV, thus bringing into, question and challenging the previously reconstructed history of BSMV and the hypothesis of a recent origin of the virus. Similarly, ancient plant and soil samples could also be analyzed by NGS for plant viruses and viroids, as illustrated by the recent discovery of viral genomes in 700-year-old caribou feces from a subarctic ice patch (Ng et al., 2014) and of a giant DNA virus, Pithovirus sibericum, in a 30,000-year-old Siberian permafrost sample (Legendre et al., 2014). Such analyses would allow a significant gain in knowledge of the evolutionary history of plant viruses and viroids over the past few millennia, a time period hypothesized to have seen the emergence of several very important viral genera, as, for example, the evolutionary radiation of the potyviruses (Gibbs et al., 2008), the most important plant virus genus.

1.11 Resistance to virus infection

Resistance to or control of plant virus diseases may be divided into natural and genetic resistance and approaches. Traditional resistance breeding (i.e., using resistant genes from other cultivars or related species), cross-protection, use of antiviral substances of plant origin, resistance to viruses via resistance to vectors, as well as other traditional protection strategies are beyond the scope of this review. In this section, we will deal with major biotechnological resistance strategies that had a significant impact on developing plants resistant to viral infections.

1.11.1 Pathogen-derived resistance in transgenic plants

The first successful demonstration of genetically engineered resistance to a plant virus was reported by Abel et al. (1986), who found that transgenic tobacco plants expressing the CP gene of TMV displayed a significant delay in symptom development following inoculation with the virus. These findings indicate that plants can be genetically transformed for resistance to virus. This form of viral CP-mediated resistance, in which a sense RNA encoding the CP is expressed, was demonstrated for a number of different viruses. Other viral-coded proteins such as RdRp and movement proteins in transgenic plants also develop resistance to viral infection. Some commercial plants such as papaya plants resistant to infection by papaya ring spot virus in Hawaii have been commercialized successfully (Gonsalves, 1998). The pathogen-targeted transgenic resistance may also include antiviral ribozymes, antisense RNAs, satellite RNA–mediated resistance, antibody genes, pokeweed antiviral protein genes, and multiple gene strategy (Hadidi et al., 1998).

1.11.2 RNA silencing

RNA silencing is an important mechanism of gene regulation in many organisms, including plants, which is induced by dsRNA. The process can be divided into RNA-mediated transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS). Lindbo and Dougherty (1992) were the first to show that in tests on the tobacco etch virus resistance conferred by the virus-derived transgenes, an untranslatable transgene derived from the CP gene of the virus made tobacco plants tolerant of the virus infection. In these plants, viral RNA was rapidly degraded and kept at a subnormal level (Lindbo et al., 1993), an effect similar to that of PTGS. The roles of RNA silencing in regulating virus replication and cross-protection between virus strains were revealed (Covey et al., 1997; Ratcliff et al., 1997).

Viruses and viroids have the ability to induce both types of RNA silencing in infected plants. RNA silencing is a cell-surveillance system that recognizes dsRNA and ssRNA with a compact secondary structure; it specifically inactivates RNA viruses and viroids (by PTGS) as well as DNA viruses (by TGS and/or PTGS), using small interfering RNAs (siRNAs) as a guide (for review, see Barba and Hadidi, 2009; Sano et al., 2010; Wang et al., 2012; Flores et al., 2015; Zhang et al., 2015; Dadami et al., 2017). Sources of dsRNA include replication intermediates of viruses/viroids, transcription of inverted repeats, stress-induced overlapping antisense transcripts, and RdRp transcription of aberrant transcripts (Wassenegger and Krczal, 2006). The dsRNA trigger is cleaved into 21–24 nucleotide duplex siRNAs, by a ribonuclease III (RNAse III)–like enzyme termed Dicer in animals and Dicer-like in plants (Bernstein et al., 2001; Hamilton and Baulcombe, 1999; Zamore et al., 2000). The formation of functional RNA–induced silencing complexes involves retention of one, the siRNA guide strand, and release of the other, the siRNA passenger strand (Kim, 2008). Importantly, besides transcript degradation, siRNAs can also mediate translational arrest (Brodersen et al., 2008).

1.11.3 Genome editing

Genome editing is an approach in which a genome sequence is directly changed by adding, replacing, or removing DNA bases. A nuclease is directed to the desired genome location, where a DNA break is introduced; different types of nucleases have been developed for this purpose. All nucleases are composed of two components: the major component is the nuclease itself (the catalytic domain), which cleaves the DNA, while the secondary component recognizes and binds DNA in a sequence-specific or nonspecific manner. The major class of genome editing that is now being used by many laboratories, as it has been used in plant virology is the clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas system, which uses a nuclease called Cas9 to introduce a dsDNA cleavage (Doudna and Charpentier, 2014). Unlike the genome editing system, zinc finger nucleases or transcription activator–like effector nucleases (Gaj et al., 2013), this system does not use a protein-based DNA recognition domain. The Cas9 nuclease is guided to the target DNA binding site by an RNA sequence that is designed to precisely bind to a complementary DNA sequence, allowing for the Cas9 nuclease to make a specific cut. Thus, with CRISPR-Cas, a synthesized guide RNA is needed. Additional advantages of the CRISPR-Cas system over the other genome editing systems are that (1) multiple genomic sites may be edited at once; (2) only a few manipulating tools are required, making it faster and easier to use; (3) it is not species-specific, as some other genome engineering systems are; and (4) it can be used to manipulate a number of different genes at one time. Moreover, the ability to redirect the dsDNA targeting capability of CRISPR-Cas9 for RNA-guided single-strand RNA binding and/or cleavage (which is denoted RCas9, an RNA targeting Cas9) has been shown (Abudayyeh et al., 2016; East-Seletsky et al., 2016; O’Connell et al., 2014). The nuclease Cpf1 has been reported as an alternative to the Cas9 enzyme (Zetsche et al., 2015). CRISPR-Cpf1 is smaller in size than CRISPR-Cas9 and it was documented to make genome editing easier and more precise. CRISPR-Cas13a (formerly C2c2; Abudayyeh et al., 2016) has been shown to engineer interference with turnip mosaic virus, a potyvirus, in N. benthamiana (Aman et al., 2018). When Cas13a is combined with CRISPR RNA (crRNA), it forms a crRNA-guided RNA-targeting CRISPR effector complex (Abudayyeh et al., 2016).

1.11.4 CRISPR-Cas system editing confers resistance to plant viruses

DNA geminiviruses: Current conventional and molecular strategies of controlling DNA geminiviruses have met with marginal success (Lapidot et al., 2015; Reyes et al., 2013; Yang et al., 2014). The application of the CRISPR-Cas9 to geminiviruses has been shown to enhance resistance to tomato yellow leaf curl virus (genus Begomovirus) in N. benthamiana (Ali et al., 2015), bean yellow dwarf virus (genus Mastervirus) in N. benthamiana (Baltes et al., 2015), and beet severe curly top virus (genus Curtovirus) in N. benthamiana and Arabidopsis (Ji et al., 2015). Ali et al. (2015) were also successful in enhancing N. benthamiana resistance to three different geminiviruses simultaneously.

RNA viruses: Recessive plant genes, such as those coding for translation initiation factors, have been reported to confer host resistance to infection by RNA viruses (Lellis et al., 2002; Truniger and Aranda, 2009; Sanfaçon, 2015). The translation initiation factor eIF4E and its isoform have been shown to interact with the small VPg protein of members of the families Potyviridae and Secoviridae and of the genus Sobemovirus in infected plant cells. Disrupting the covalent linkage of VPg to the viral RNA 5′-terminus by mutagenesis or silencing interferes with virus infectivity (Sanfaçon, 2015). The CRISPR-Cas9 system has been used successfully to target two sites of eIF4E gene function in cucumbers to develop resistance to infection by three positive-strand RNA viruses, namely cucumber vein yellowing virus (family: Potyviridae, genus: Ipomovirus), papaya ring spot virus-w, and zucchini yellow mosaic virus (family: Potyviridae, genus: Potyvirus) (Chandrasekaran et al., 2016). Complete resistance in Arabidopsis to turnip mosaic virus (genus: Potyvirus) using a CRISPR-Cas9 system targeting the plant eIF (iso) 4E gene has also been reported (Pyott et al., 2016). CRISPR-Cas13a (formerly C2c2; Abudayyeh et al., 2016) has been shown to engineer interference with turnip mosaic virus, a potyvirus, in N. benthamiana (Aman et al., 2018). When Cas13a is combined with crRNA, it forms a crRNA-guided RNA-targeting CRISPR effector complex (Abudayyeh et al., 2016).

Viroids: No plant genes have been reported to confer host resistance to infection by viroids. Thus, conventional plant breeding has not been used for producing varieties resistant to viroids. Moreover, since stable and durable resistance to viroid infection has not been found in most viroid host species, different molecular strategies have been applied to introduce viroid resistance in these plants (Dalakouras et al., 2015; Kovalskaya and Hammond, 2014; Hammond and Kovalskaya, 2017). These strategies have been partially successful, and new molecular approaches for controlling viroid diseases by genome editing of viroid RNAs using CRISPR-Cas9 or CRISPR-Cpf1 systems could be useful, as they have been in controlling plant viruses (Hadidi et al., 2016). Because the genome information in viroids is very much compressed as a consequence of their small size, most artificial mutations are expected to be deleterious, particularly when affecting a critical function. By the way of example, Hadidi and Flores (2017) highlighted in three representative viroids, PSTVd, avocado sunblotch viroid, and peach latent mosaic viroid, some functionally relevant motifs that could be specifically targeted by the CRISPR-Cas9 system (Hadidi and Flores 2017), the CRISPR-Cas13a system, or both (Hadidi, 2019).

1.12 Control by exclusion

Natural spread of viruses or viroids by insects, pollen, or seeds is difficult to control. However, preventing pathogen spread by human movements is desirable and exclusion of viruses or viroids from a country, state, region or an area is possible and potentially under national and international control. Exclusion may be defined in local terms for pathogens that are already established in an area within a country. The legal requirements necessary for exclusion are commonly known as quarantine or phytosanitary regulation. The cost of exclusion is significantly less than eradication or significant losses year after year.

Exclusion requires that viruses or viroids to be eliminated from propagating material, thus preventing them from being distributed in planting material. It was from this concept that certification schemes were developed in the first quarter of the 20th century and have been used in many countries to produce stocks of a defined health status that also met quality standards. Thus, phytosanitary certification is a system whereby countries have an obligation to ensure that exported plants are free of pests of concern to the importing country (Barba and James, 2017). Elimination of viruses or viroids can be achieved by different techniques such as thermotherapy which is performed either in vivo or in vitro, cold therapy, meristem tissue culture, a combination of in vitro therapy and meristem tissue culture, in vitro micrografting, cryotherapy and in vitro chemotherapy (Barba et al., 2017; Laimer and Barba, 2011). Regulations have been frequently modified in the light of advancements in virus or viroid detection and diagnosis. A batch of seed-potatoes is certified only if it meets the standard of a grade. If not, it is to be used for direct consumption only (De Bokx and Van der Want, 1987). The principles of the production of prime seed and other planting materials apply to various other crops.

Quarantine or phytosanitary regulation are overseen by governments on the basis of agreements produced by international organizations (Barba and James, 2017; Reed and Foster, 2011). Quarantine is considered a primary strategy to prevent the introduction of pests in areas that are otherwise free of the target plant pathogens; this involves some form of isolation of the imported germplasm and pathogen testing (Barba and James, 2017). Reed and Foster (2011) explained the international quarantine regulations by stating: International quarantine regulations are overseen mainly by the ‘Agreement on the Application of Sanitary and Phytosanitary Measures [SPS] 1994,’ under the World Trade Organization ‘General Agreement on Tariffs and Trade’ (the WTO-SPS). The SPS agreement identifies the International Plant Protection Convention (IPPC), administered by a Commission under the United Nations Food and Agriculture Organization (FAO), to promote international cooperation in controlling pests and providing international standards. The WTO-SPS, to which most countries are signatories, requires that quarantine measures are based on scientific principles specified in a risk assessment, are nondiscriminatory, and must take into account appropriate levels of protection, consistency, inspection procedures, treatments, pest prevalence, and pest-free areas (IPPC, 2006). They must also be publicly available and