Keywords

1 Introduction

In the natural environment, plants are colonized by a microbiota (a microbial community) composed of various organisms including viruses, bacteria and fungi. The majority are not pathogenic and do not harm their host. On the contrary, they are necessary for plant wellness and participate in a mutualistic interaction beneficial to themselves and their plant host. For instance, some plant viruses confer tolerance to stress, such as heat or drought stresses (Marquez et al. 2007; Xu et al. 2008), allowing the infected plants to colonize extreme environments (such as the extremely hot soil in the Yellowstone Park, US) and to survive sudden changes in their habitat (such as water fluctuations due to tide surges).

Viruses are ancient microbes and shape the evolution of all living organisms by their host:virus interactions and by transferring genetic material among species. Today many viruses are used for beneficial applications including in medicine to fight bacterial infections, in forest ecosystems to kill defoliating caterpillars, and in nano/biotechnology as carriers for genetic information, delivering drugs into the right type of cells or expressing proteins in plants, insect cells or bacteria. In contrast, and this is true especially in the agricultural landscape, some plant viruses, as well as bacteria and fungi, can cause diseases that become eventually lethal to their hosts, and most importantly can cause a substantial yield reduction in crops that are important for food and fiber. Deleterious viruses seem to have evolved as consequence of the advent of agriculture, and are relatively evolutionarily new, compared to viruses found in natural landscapes. The negative impact of plant viral diseases, even to the point of limiting food supply, is the reason we are concerned about growing healthy crops. It is estimated that plant diseases in general cause up to a 14 % loss in total crop production every year, a percentage that equals hundreds of billions of US dollars in lost revenues. Ten to 15 % of this loss can be attributed to viruses, but for specific crops and in specific locations, like in the Asian and African continents where food supplies are already limited, the losses can have severe direct effects on human health. To control viral diseases, we routinely use a variety of strategies, ranging from the application of pesticides aimed to reduce the number of insect vectors that spread viruses, the adoption of Integrated Pest Management (a system designed to use multiple pest management practices in an environmentally sound manner), the establishment of plant and animal quarantine areas, the use of certified germplasm material (a collection of genetic resources, for instance seeds and tubers) and various forms of genetic resistance.

2 What Are Plant Viruses, and Do Plant Viruses Differ from Animal Viruses?

Plant viruses are generally smaller and less complex than animal and bacterial viruses. The sizes of the viral particles range from ~20 to 200 nm, and their chemical composition is generally simple. Virus particles typically have an outer shell made of proteins arranged in a geometrical form, either in a rod-shaped or in an isometric “coat” (Fig. 13.1).

Fig. 13.1
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Representative plant virus particles purified from infected leaves. (a) Tobacco streak virus (isometric). (b) Pea seed-borne mosaic virus (rod shaped)

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The viral shell is composed of repetitive small protein subunits and for some plant viruses the shell is surrounded by a lipid (fatty) membrane derived from the host cell. The viral genome is protected inside the shell, and can be made of either RNA or DNA (Fig. 13.2).

Fig. 13.2
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(1) The capsid is the protein shell that encloses the nucleic acid. It is built of structural subunits. (2) Coat protein subunits are the smallest functional equivalent building units of the capsid. (3) The capsid with its nucleic acid is called the nucleocapsid. (4) The nucleocapsid may be protected by an envelope. (5) The virion is the infective virus particle

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The viral genome can be circular or linear, can be composed of one or more “chromosomes” and can be contained in single or multiple viral particles or virions. The plant viral genome size is generally small, usually ranging from 3,000 to 30,000 nucleotides (blocks that build RNA and DNA), while animal virus genomes can consist of 800 thousand nucleotides and encode for up to a few hundred proteins. The virions of a few viruses may contain viral proteins necessary to initiate viral replication or multiplication, but viruses are unable to replicate without the host’s cellular machinery; they are intracellular molecular obligate parasites. They exploit and highjack the host cell to multiply and they cannot perform this outside living cells. Since plant viruses are so small, they can express only a few proteins, and are thus amazing in their ability to replicate and respond to plant defenses, considering their limited genomic arsenal. Viruses rely on multifunctional proteins, protein modifications and on timely regulated protein expression and genomic replication to successfully complete their life cycle.

2.1 Can Plants Defend Themselves Against Viruses?

Let’s have a first look at how plant defenses function, and how they differ from animal defenses.

Plants are multicellular organisms and their cells are interconnected by channels called plasmodesmata. Plasmodesmata serve as freeways that allow small molecules to travel between cells, or even throughout the entire plant. Plant viruses take advantage of the plasmodesmata to spread within the infected plant (Fig. 13.3).

Fig. 13.3
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Plant cells are protected by cell walls, and connected by plasmodesmata

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Plants possess constitutive and inducible defenses against insects, microbes and other stressors (Dangl and Jones 2001; Howe and Jander 2008). Constitutive defenses are always present; they include physical barriers such as modified cell wall composition or the presence of leaf trichomes (hairs or appendages), and chemical barriers such as production of chemical deterrents. Inducible defenses are those that are ‘turned on’ only as consequence of pathogen attack, when the plant cell’s innate immune system recognizes pathogen-encoded effectors that are signature molecules specific of a class of organisms. Hypersensitive Response, Reactive Oxygen Species release and Programmed Cell Death are the names given to plant reactions linked to Resistance (R)-gene mediated resistance. Here upon recognizing the pathogen, plant cells release chemical defenses and “commit suicide”, in the attempt to contain infections from spreading into healthy tissues. The leaves of plants expressing these reactions show necrotic spots, corresponding to the points in which the attack started (Fig. 13.4).

Fig. 13.4
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Pathogens are perceived by plants and activate local responses as well as systemic responses in the attacked plant

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Signals originated from the site of infection then stimulate the Jasmonic Acid, Ethylene and/or Salicylic Acid dependent or independent defense pathways in distal parts of the plants (Fig. 13.5). These pathways are sequential events that turn on and off other genes and their products to help the plant in its fight against pathogens. Viruses and other plant pathogens have evolved means to counteract plant defeneses, thus plant resistance and pathogen counter defense represent an ongoing evolutionary “arms race”.

Fig. 13.5
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Pathogen infection, cell necrosis, or insect wounding activate the salicylic acid or the jasmonic acid and ethylene pathways, leading to local and systemic plant responses. These defense pathways are interconnected

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Finally, plants lack the somatic adaptive immune system (antibodies) typical of animals and do not possess lymphocytes (type of white blood cells).

2.2 Are Cultivated Plants More Susceptible to Viruses Than Their Wild Relatives?

Individual plants in a natural population are not genetically identical and usually differ in their resistance to pathogens. By contrast agricultural species grown in monocultures (where all the plants belong to the same species and are essentially identical) are equally susceptible or resistant to specific pathogens. Thus, if wild relatives that show degrees of resistance or tolerance to the same pathogen can be identified, these can serve as sources of genetic resistance for cultivated crop plants. Resistance can be conferred by expression of a single host gene (R-gene), or by multiple genes, and plant breeders try routinely to introgress (move) genes for resistance found in wild plants into cultivated varieties via traditional breeding, or via transgenesis.

2.3 Examples of Natural Resistance

Many plants show R-gene mediated natural resistance to viruses. For example, in many Arabidopsis thaliana accessions (a collection of plants from the same location), some proteins produced by the plant called the Restricted Tobacco etch virus Movement RTM1, RTM2 and RTM3 restrict long distance movement of plant viruses called potyviruses. This resistance is not present in A. thaliana accessions showing amino acid changes in their RTM proteins. Since the viral coat protein is the effector recognized by the RTM proteins, potyviruses that naturally show changes in their outer shell structure are not recognized by A. thaliana without changes in their RTM proteins and can infect plants with Rtm genes.

Many tomato (Solanum) species show natural resistance to viruses called tospoviruses, as well as to other plant pathogens. S. peruvianum, S. chilense, S. habrochaites and S. pimpinellifolium are used as source of resistance genes to be introgressed into cultivated tomatoes (Solanum lycopersicum) that are susceptible to tospoviruses. A single gene called Sw-5b introgressed from S. peruvianum into S. lycopersicumcultivar Stevens shows broad-spectrum resistance to tospoviruses. The Sw-5b gene belongs to a particular class of plant resistance genes and is similar to other resistance genes such as the tomato nematode and aphid resistance gene Mi. The resistance conferred by Sw-5b can elicit a hypersensitive response in virus inoculated tissues, where it blocks the spread of the virus, but the resistance is absence in tomato fruits.

Some plant proteins called Endosomal Sorting Complex Required for Transport (ESCRT) are involved in endosome maturation. Endosomes are cellular vesicles that help in transporting proteins to different destinations in the cells and to or from the cell surface. Most viruses exploit the ESCRT system during their replication and movement, and impairment in ESCRT interferes directly with the ability of the viruses to replicate and move. In fact, Arabidopsis plants modified to lack ESCRT show inhibited viral replication and infection for a group of viruses called tombusviruses.

The resistance to a virus called Tobacco mosaic virus in the plant Nicotiana glutinosa is due to the ‘Necrotic-type response to infection with TMV’ N gene. This gene product also interferes with viral replication and as the name suggests induces plant cells to necrotize (commit suicide) in order to stop viral infection.

2.4 Examples of Transgenic Resistance

In absence of natural resistance, today we can sometimes use transgenesis to incorporate viral resistance traits into crop plants. In some instances, we can move natural R-genes from one crop to another crop, but this is not always effective, probably due in part to the genetic background of the recipient crop plant. In the late 1980s, scientists started exploring the idea that inserting viral genes into plants could trigger the transgenic plants to become ‘immune’ to viruses, in a kind of self-perpetuating plant vaccination. In 1986 Powell-Abel et al. successfully generated the first transgenic tobacco plants expressing the TMV coat protein. In 1988 Nelson et al. engineered whole transgenic tomatoes to express the TMV coat protein. Some of the transgenic lines were partially resistant to TMV infection. Since then, many plants (barley, canola, corn, oat, rice, wheat, chrysanthemum, dendrobium, gladiolus, grapefruit, grapevine, lime, melon, papaya, pineapple, plum, raspberry, strawberry, tamarillo, walnut, watermelon, alfalfa, sugarcane, bean, clover, groundnut, pea, peanut, soybean, lettuce, pepper, potato, squash, sugar beet, sweet potato, and tomato) have been transformed to be resistant to one or more viruses. However, of all the plants that have been generated and tested in laboratory or greenhouse settings for their viral resistance, few have reached the market.

The transgenic summer squash line ZW-20 resistant to Watermelon mosaic virus and Zucchini mosaic virus, and the transgenic squash line CZW-3 resistant to Cucumber mosaic virus, Watermelon mosaic virus and Zucchini mosaic virus were commercially released in the US in 1994 and 1996, respectively (Tricoli et al. 1995). A transgenic papaya (Gonsalves et al. 1997) resistant to Papaya ringspot virus (PRV) was released in 1998, while transgenic green pepper varieties and tomato resistant to Cucumber mosaic virus are today released in the People’s Republic of China. Two potato lines resistant to Potato virus Y were deregulated in Canada (1998) and the US (1999) but were later abandoned because of the extremely negative public opinion (Kaniweski and Thomas 2004). Of the transgenic plants released to the market, the most successful story comes from the use of PRV transgenic papaya in Hawaii. This event saved the papaya industry in Hawaii from complete destruction due to PRV, allowed the cultivation of non-transgenic papaya cultivars in between transgenic fields, and increased the cultivar diversity in the islands. Today transgenic papaya cultivars resistant to local PRV strains have been developed in Thailand, Jamaica, Brazil, and Venezuela and are at different stages of deregulation.

The latest virus resistant transgenic plant that has been deregulated (2011) in the US is the Plum pox virus resistant ‘HoneySweet’ plum. Research to establish the safety and characteristics of this plum variety took more than 20 years, and it has been particularly important since there is no high level of PPV resistance known in Prunus domestica, P. spinosa and P. insitita. Only P. cerasifera offers a cultivar that is hypersensitive to PPV inoculation, and young plants of this cultivar naturally die when exposed to the virus. ‘HoneySweet’ plums score high in fruit quality and yield, and are today crossed with other plum varieties since they can transmit the dominant resistant trait as a single locus. Scorza et al. in 2013 wrote a highly remarkable review on the process of deregulation on HoneySweet.

The mechanism that allows these transgenic plants to be resistant to viruses is not always known, but in most of the cases the resistance is due to a host natural defense system called gene silencing or RNA interference.

3 RNAi: A Newly Discovered, Nucleic Acid Sequence-Based Inducible Defense Mechanism

The eukaryotic inducible defense mechanism that evolved specifically against viruses, called RNA interference (RNAi), or gene silencing (Voinnet 2001; Waterhouse et al. 2001), works against specific nucleotide sequences, and it is thus atypical when compared to the classic effector-mediated plant defense system described above.

3.1 How Does RNAi Work?

When viruses replicate in eukaryotic cells, a double-stranded RNA (dsRNA) form of the viral genome is produced by the viral enzyme RNA dependent RNA polymerase that uses the RNA genome from the virus as a template, and copies it in an RNA molecule of opposite polarity. Since the two RNA molecules are complementary, they can anneal to each other and form a double stranded RNA helix. DsRNA can also be generated from the pairing of complementary stretches of RNA on the same molecule, and is not always linked to viral replication. Large dsRNA molecules, such as those generated during virus infections or viral replication, are not found in healthy cells and their presence is recognized as foreign and serves as the trigger of the eukaryotes’ (e.g. plants) RNAi pathway.

When dsRNA is found in a healthy plant cell, a plant enzyme called Dicer cleaves the dsRNA into small duplex fragments that are 21 nucleotides long, called small interfering RNAs (siRNAs). Dicer has a pocket that is exactly 65 Å in size, the distance that equals 21 nucleotides (Fig. 13.6). This pocket serves as molecular ruler. The siRNA duplex is dissociated into two strands by Dicer and one of the two strands (the sense or passenger strand) is degraded by the same enzyme. The second strand (the antisense or guide strand) gets incorporated into an enzymatic complex called the RNA Induced Silencing Complex, or RISC. RISC uses the antisense strand as template to find and hybridize with RNA from viruses and with a complementary nucleotide sequence (more viral RNA), and to degrade it. In this way, cells are able to find and destroy viral RNA and to distinguish it from other cellular messenger RNA (mRNA).

Fig. 13.6
figure 6

After viral infection, viral dsRNA is produced in eukaryotic cells. The dsRNA is recognized by the cell enzyme Dicer and processed into 21 nucleotide long siRNA molecules that are incorporated into the cell RISC complex and used to search for complementary viral RNA sequences for their degradation. Red and blue colors are used to show complementary and opposite RNA polarities

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Messenger RNA (mRNA) is the nucleic acid that is transcribed using DNA as template. RNA leaves the nucleus and is translated into proteins in the cell cytoplasm.

While animals have evolved other defenses such as an interferon based signals to alert healthy cells about a virus attack, and antibodies to recognize specific viruses and other microbes, plants especially, but also insects, rely heavily on the RNAi pathway to defend themselves against viruses.

3.2 How Can We Manipulate RNAi to Induce Virus Resistance in Plants?

If we genetically transform plants to express double-stranded RNAs, the plant RNAi machinery will recognize the dsRNA and initiate a response. If the engineered plant expresses a plant virus double-stranded RNA sequence, the plant RNAi response will recognize that viral RNA. This strategy has been used to develop virus “immune” plants: plants that recognize viral nucleic acids produced during viral infection or replication, degrade those viruses, and thus stop the infection or viral replication process.

So, going back to the examples of the transgenic plants expressing virus sequences such as those encoding for the viral coat protein, those plants are very efficient in recognizing and destroying the invading virus RNAs. If we analyze the genome of those transgenic plants, we can find the inserted viral sequence, and the corresponding 21 nucleotide siRNAs derived from the inserted sequence but resulting from the Dicer activity (Fig. 13.7).

Fig. 13.7
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Transgenic plants can be made by inserting a part of a viral sequence and its complementary sequence, under the control of a plant or virus promoter. The plant will express a double stranded form of the viral sequence that will trigger the plant RNAi pathway against those specific viral sequence. If the plant is challenged by the target virus also containing that particular sequence, the plant will use the primed RNAi pathway to destroy the viral RNA and halt infection

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Other transgenic plants produced in laboratory trials where RNAi against viruses has been exploited are: walnut, ryegrass, tomato, tobacco, sweet potato, soybean, potato, rice, poplar, opium, maize, ornamental crops, and apple.

3.3 Does RNAi ALWAYS Involve the Use of Transgenic Plants?

In addition to transgenic methods to enable plants to utilize RNAi, ‘Viral induced gene silencing’, or VIGS is used to introduce specific nucleotide sequences that can induce RNAi effects in plants via non-lethal recombinant viruses. These viruses are used as carriers to express the nucleotide sequence complementary to the one that we wish to target via RNAi, but since these viruses do not integrate into the plant genome, the resulting plants are not transgenic. These viruses are usually inoculated (or transferred) mechanically into their host plants. A type of VIGS is for example used in Brazil, Australia, South Africa and Japan to fight Citrus tristeza virus (CTV) in citrus orchards. There, a mild strain of CTV is inoculated in the trees, and serves as vaccine against severe CTV strains (Costa and Muller 1980) (Fig. 13.8).

Fig. 13.8
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Plant on the far left is a healthy citrus plant. Plant on the far right is infected with a severe CTV strain (depicted by a blue leaf) and plant in the middle left is infected with a mild CTV strain (red leaf). If a plant is inoculated first with a mild CTV strain (middle right) and then by a severe CTV strain (red and blue leaf respectively), the plant will be partially protected and will grow better than plants infected by the severe CTV strain

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3.4 Issues Linked with VIGS and RNAi

There are a few issues associated with the use of RNAi against plant viruses, due to the ability of plant viruses to evade the plants RNAi response. Plant viruses evolve rapidly and can mutate their nucleotide sequences. If their nucleotide sequence changes compared to the one targeted by RNAi, it may not be recognized anymore by the RISC complex. New strains of viruses with differences in the RNAi target region are always emerging, and would not be affected by RNAi. Third, plant viruses encode for proteins that can allow the virus to evade the RNAi machinery. These proteins are called ‘suppressors of gene silencing’ (Qu and Morris 2005), and other viruses expressing potent suppressors of gene silencing can sometimes protect the viruses that are the RNAi target, if they co-infect the transgenic plant. Mixed infections, where multiple viruses infect the same plants, are common in nature.

3.5 Modification of the RNAi Strategy: RNAi, or Gene Silencing, Can Be Used, for Instance, to Affect Insect Vector Performance

Viruses in nature can be either vertically transmitted via infected pollen and seeds, or horizontally transmitted by virus vectors, most commonly insects (Nault 1997). Some viruses can be transmitted in both ways, but often agriculturally significant viruses are transmitted mainly by their insect vectors. Viruses are picked up by insects when they feed on virus-infected plants and then are introduced into healthy plants when the insects move from plant to plant and eject saliva containing viruses in the newly encountered plants, while feeding.

Novel research focuses on provoking the plant to control insect vectors and their associated viral diseases using RNAi, either through genetic transformation or VIGS. Many studies suggest that RNAi effects can be induced in insect cells, and even in whole insects that feed on such plants (plants expressing the RNAi sequences by transformation or VIGS). Artificial dsRNAs can be used to trigger the RNAi pathway. If the artificial dsRNA nucleotide sequence is synthesized (artificially assembled) to be identical to a specific insect mRNA, then that mRNA becomes the target of the RNAi machinery for destruction, effectively ‘silencing’ the corresponding gene and stopping protein translation. For instance, if a plant is transformed to produce a dsRNA molecule whose sequence corresponds to an insect gene, the plant will produce siRNAs that will target the insect RNA target, and the plant might become insect and virus resistant. How? When insects feed on the transgenic plants, they ingest the plant-produced siRNAs, and since insects also have a RNAi machinery, their defense system will use the ingested siRNAs to find and destroy the corresponding target RNA sequence, in this case an insect RNA!

3.6 Modification of the RNAi Strategy: RNAi, or Gene Silencing, Can Be Used, for Instance, to Affect Insect Vector Performance

A recent study (Baum et al. 2007) has reported the use of RNAi in corn roots to control the western corn rootworm. The transgenic corn plants express in its roots the dsRNAs against the western corn rootworm ATP-ase mRNA, (ATP-ase is expressed in the insect gut and necessary for many vital processes) and these plants have been shown to be highly resistant to rootworm damage. This technology can be used, for instance to increase the durability of transgenic corn using the Bt (Bacillus thuringiensis) resistance.

4 Future Perspectives

Transgenesis in plants and the use of RNAi technologies is a subject of hot debates. Recently, scientists have discredited (Dickinson et al. 2013) a study published by Zhang et al. in 2012 where the authors reported plant microRNA168 in blood of mammals (humans and mice) fed on rice, and that this microRNA regulated mammalian gene expression in the liver. MicroRNAs belong to a class of small RNA molecules very similar to the one composed by siRNAs. MicroRNAs are produced by every organism and are used to regulate gene expression, especially during the organism’s growth and development. Since the structure of miRNAs is similar to the one of siRNAs, this study indirectly poses a question mark on the stability across the mammalian digestive system of siRNA generated by transgenic plants and, not surprisingly, is the subject of intense debate. SiRNAs seem to be stable in the digestive tract of arthropods and siRNAs generated by plants and ingested by insects in some cases have been shown to affect distant organs, but no study has proven the same stability in mammalians. At the same time, we are exposed every day to our own miRNAs and ingest miRNAs produced by plants, other animals and even by microorganisms in large amounts. Further studies are needed to examine the stability and potential effects of miRNAs in the mammalian digestive system.