Plant Viruses and Technology

 Chapter 15

Plant Viruses and Technology SUMMARY The development of DNA technology over the last 30–40 years has provided a wide choice of opportunities for using nucleic acids and viruses for experimental, pharmaceutical, and industrial purposes. The relatively small size of their genomes and virus particles has enabled a detailed understanding of how they function, and this knowledge is now being used for an increasing number of purposes ranging from field resistance against viral pathogens to functional genomics of plants and pharmaceutical and industrial processes. In this chapter, I describe some of the uses to which viruses are being put.

Viruses are very amenable to manipulation providing a wide range of uses both in fundamental studies such as molecular functions in plants and in applied aspects such as conferring virus resistance in crop and industrial and pharmaceutical processes.

I.  TRANSGENIC PROTECTION AGAINST PLANT VIRUSES A. Introduction It is now possible to introduce almost any foreign gene into a plant and obtain expression of that gene. In principle, this should make it possible to transfer genes for resistance or immunity to a particular virus, across species, genus, and family boundaries. Furthermore, genes can be designed to interfere with directly, or induce the host to interfere with, the virus replication cycle. Several approaches to producing transgenic plants resistant to virus infection are being actively explored. There are essentially three sources of transgenes for protecting plants against viruses: (i) natural resistance genes; (ii) genes derived from viral sequences, giving what is termed Pathogen-Derived Resistance (PDR); and (iii) genes from various other sources that interfere with the target virus. These will be discussed in the following sections. There have been numerous reviews on the subject including Bonas and Lahaye (2002), Morroni et al. (2008), Prins et  al. (2008), Gottula and Fuchs (2009), and Reddy et al. (2009).

Plant Virology, Fifth Edition. © 2014 2012 Elsevier Inc. All rights reserved.

B.  Natural Resistance Genes Molecular aspects of genes found in plant species that confer resistance to various viruses are discussed in Chapter 11. When a resistance gene has been identified, it can be isolated and transferred to another plant species. The Rx1 gene that gives extreme resistance to potato virus X (PVX)1 has been isolated from potato and transformed into Nicotiana benthamiana and N. tabacum (Bendahmane et  al., 1999), where it gives resistance to the virus. Similarly, the N gene, found naturally in N. glutinosa, and that confers hypersensitive resistance to TMV, gives resistance to TMV when transferred to tomato (Whitham et al., 1996). The application of new genomic, bioinformatic, and molecular techniques to understanding R gene functions is opening possibilities for manipulating the system to provide new sources of resistance (Campbell et al., 2012; Gururani et  al., 2012). Among the possibilities is to use new R genes that are being discovered, modifying existing R gene to give novel resistances and manipulating the resistance pathway. As discussed in Chapter 11, Section III much of the specificity of R gene is determined by the leucine rich repeat regions. These regions can be manipulated in vitro to give yet further sources of resistance.

C.  Pathogen-Derived Resistance The ideas leading up to the concept of PDR for plant viruses were first postulated by Hamilton (1980) and are encapsulated as a general concept by Sanford and Johnson (1985). They suggested that the transgenic expression of pathogen sequences might interfere with the pathogen itself terming this concept “parasite-derived resistance.” Since then, several names have been used for this approach including “non-conventional protection,” “transgenic resistance” or “engineered virus resistance,” but PDR is now the generally accepted term. Since the mid-1980s, this approach has attracted major interest and is the main one by which transgenic protection is being produced against viruses in plants. The first demonstration of PDR against plant viruses was by 1

Acronyms of virus names are shown in Appendix D

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Powell-Abel et  al. (1986) who showed that the expression of TMV coat protein (CP) in tobacco plants protected those plants against TMV. This opened the floodgates for extensive research both on protecting crop species against viruses and on the mechanisms involved. The basic idea arising out of Sanford and Johnson’s concept is that, if one understands the molecular interactions involved in the functioning of a pathogen, mechanisms can be devised for interfering with them. Although this concept applies to all pathogens and invertebrate pests, it has mainly been used against viruses because of their relatively simple genomes. In developing the concept, it was recognized that the interactions of interest occur at all stages of the virus infection cycle and that they can potentially be interfered with in various ways, for example, by blocking the interaction or by decoying one or more of the molecules involved in the interaction. This then led to the idea that the overall strategy as being one of attacking specific viral “targets” with specific molecular “bullets.”. Some examples of “targets” and attacking mechanisms or “bullets” are given in Table 15.1. However, in practice, much of the development of this approach was done without detailed knowledge of the precise molecular mechanisms involved, and analysis of these results has thrown light on several new mechanisms. Perhaps the most important is the gene silencing phenomenon described in Chapter 9, Section I and which is further discussed in Section I, C, 2 below. In this rapidly expanding subject, there are various terminological problems. The main one, whether to term this phenomenon resistance or protection, is discussed in Section I, E. I will use the term protection wherever possible, but in situations where it has been used widely (such as PDR), I will keep the term resistance. Currently, there are two basic molecular mechanisms by with PDR is thought to operate. In some systems the expression of an unmodified or a modified viral gene product interferes with the viral infection cycle—this I will term protein-based protection. The second mechanism does not involve the expression of a protein product and I will call this nucleic acid-based protection.

1.  Protein-Based Protection As noted above, the first demonstration of PDR involved the expression of TMV CP (Powell-Abel et  al., 1986). Since then, there have been many examples of the use of this CP-mediated protection. The expression of other viral gene products also gives protection to a greater or lesser extent against the target virus. a.  Transgenic Plants Expressing a Viral CP The sequences encoding viral CPs are the most widely used for conferring protection in plants (Fitchen and Beachy, 1993); by 1997, CP genes from at least 35 viruses,

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TABLE 15.1  Examples of “Targets” and “Bullets” for PDR “Targets”

“Bullets”

Virus gene products

Molecular blockers

  Coat protein

  Viral gene products

 Replicase

  Mutated viral gene products

 Cell-to-cell spread function

 Antisense nucleic acid ± ribozymes

 Protease

  Sense nucleic acid  Antibodies

Control sequences  Replication control sequences Decoys    Origins of replication

 Nucleic acid control sequences

   Primer binding sites

 Satellites

 Expression control sequences

  Protease sites

   Subgenomic RNA promoters   Translational leader sequences

Other

  Splice sequences

  Nonhost resistance

From Hull (1994) with permission of the publishers.

representing 15 viral taxonomic groups, had been transformed into many different plant species (Palukaitis and Zaitlin, 1997), and there have been several other examples since then. This is because of this gene was used in the first example of this approach and because CP genes are relatively easy to identify and clone. The phenomenon is often referred to as “coat protein-mediated resistance” (CP-MR). i. Tobacco Mosaic Virus CP (reviewed by Beachy, 1999)  Bevan et  al. (1985) and Beachy et  al. (1986) first reported the expression of TMV CP in tobacco plants into which a complementary (or copy) DNA (cDNA) containing the cognate gene had been incorporated. Powell-Abel et al. (1986) showed that transgenic plants expressing TMV CP either escaped infection following inoculation or developed systemic disease symptoms significantly later than plants not expressing the gene. Plants that showed no systemic disease did not accumulate TMV in uninoculated leaves (Nelson et  al., 1987). Transgenic plants produced only 10–20% as many local lesions as controls when inoculated with a strain of TMV causing local lesions. The idea that transgenic plants resist initial infection rather than subsequent replication was suggested by

Chapter | 15  Plant Viruses and Technology

results obtained using transgenic Xanthi nc tobacco plants, in which fewer local lesions were produced than on control plants. However, the lesions that did develop were just as big as on control leaves, indicating that once infection was initiated there was no further block in the infection cycle. In transgenic plants it would be possible, in principle, for the resistance to infection to be due to the CP messenger RNAs (mRNAs) transcribed from the cDNA, to the CP itself, or to both of these molecules. To test these possibilities, Register et al. (1988) constructed a series of cDNAs generating mRNA sequences that would produce no CP, or mRNA that lacked the replicase recognition site but that would produce CP. These experiments conclusively implicated the CP rather than its mRNA in causing resistance to superinfection. Further experiments with TMV confirmed the earlier results and showed that the 3′ tRNA-like sequence was not necessary to generate resistance (Powell et al., 1990). Register et al. (1988) and Register and Beachy (1988) showed that protoplasts made from transgenic plants expressing CP were specifically protected against infection with TMV. When tobacco protoplasts took up CP, they were transiently protected from infection with TMV (Register and Beachy, 1989). Thus, CP outside the cell is probably not involved in CP-mediated protection. Pathogenesis-related proteins also do not appear to be involved in this resistance (Carr et al., 1989). ii. Dose and Sequence Dependency of Protection by TMV CP The higher the amount of virus inoculum, the lower is the protection afforded by TMV CP (Nejidat and Beachy, 1990; Bendahmane et al., 1997). There is a positive correlation between the level of protection and the sequence similarity between the transgene CP and that of the challenge virus (Nejidat and Beachy, 1990). For instance, the CPs of ToMV and TMGMV have 82% and 72% sequence identity, respectively, with that of TMV whereas that of RMV is only 45% identical; TMV CP gives much better protection against ToMV and TMGMV than against RMV. There is little or no protection against viruses from other families or genera (e.g., AMV, CMV, PVX, or PVY) (Anderson et al., 1989). Transgenic expression of TMV CP does not protect against inoculation with viral RNA (Nelson et al., 1987). iii. Other Viral CPs  Protection mediated by the transformation with the CP gene has been demonstrated for many other virus families and genera. Some examples are: Plants expressing AMV CP are also resistant to infection with AMV (e.g., Loesch-Fries et al., 1987; Tumer et al., 1987; van Dun et  al., 1987) even though this protein is required for virus replication (Box 7.3). Similar results were obtained with a mutated AMV CP (van Dun et al., 1988). Transgenic tobacco plants expressing the PVX CP gene are significantly protected against PVX infection as

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shown by a reduced number of local lesions on inoculated leaves, delayed or no systemic symptom development, and a reduction in virus accumulation in both inoculated and systemically infected leaves. The higher the level of CP expression, the higher is the level of protection (Hemenway et  al., 1988). Plants expressing an antisense CP transcript are resistant to infection with PVX, but only with low concentrations of virus in the inoculum. Other examples include, members of the family Bromoviridae CMV (Cuozzo et  al., 1988) and PDV (Raquel et al., 2008), the tobravirus TRV (Angenent et al., 1990), a member of the family Comoviridae GFLV (Valat et al., 2006), the potyvirus, PRSV, (Souza et al., 2005) and the tungrovirus RTBV (Ganesan et al., 2009). Various points can be made from these studies including: i. The extent of protection provided by such transgenic plants is correlated with the degree of expression of the CP gene. ii. The extent of protection is reduced as inoculum concentration is increased. iii. There is a variation in response of different viruses in different hosts and even between different transformation lines of a host against a specific virus. At least for PRSV in papaya such differences are related to copy number of the transgene with the number of resistant plants directly correlating with multigene copies (Souza et al., 2005). iv. CP protection appears to work with viruses that have an RNA genome (most of those listed above) and also a DNA genome virus (RTBV). v. The expressed CP does not necessarily have to be that of the target virus, but it has to be sufficiently closely related. For instance field resistance in tobacco to PVY is conferred by expression of LMV CP (Dinant et al., 1998). vi. Like TMV, the CPs of AMV and TRV do not protect against RNA inoculum (Loesch-Fries et  al., 1987; van Dun et  al., 1987; Angenent et  al., 1990), but unlike TMV, plants expressing high levels of PVX CP are resistant to infection with PVX RNA (Hemenway et al., 1988). vii. It is likely that in many other cases transformation with the CP gene does not induce any protection, for example, the ophiovirus CPsV (Zanek et  al., 2008), but most times these are not reported. Two CPs can be expressed from one construct. Marcos and Beachy (1997) designed a construct comprising the CPs of the tobamovirus TMV and the potyvirus SMV together with the highly specific TEV NIa proteinase. In plants transformed with this construct, the proteinase processes the multifunctional polypeptide to give accumulation of the two viral CPs. These plants are protected

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against both TMV and PVY. Similarly, N. benthamiana plants expressing sequences of the N gene of TSWV and the CP of TuMV are protected against both viruses (Jan et  al., 2000a). Squash lines expressing the CP genes from CMV, WMV 2, and ZYMV are resistant to all three viruses (Fuchs et al., 1998) indicating that these genes can be pyramided. Of interest is “coat” protein-mediated protection against viruses either that have several CP species or that do not have conventional capsids. Rice plants transformed with one of three structural proteins (S5, S8, or S9) of the Oryzavirus RRSV show resistance to the virus (Waterhouse and Upadhyaya, 1999) but it was not noted as to whether any of these genes were expressed as proteins. The particles of tenuiviruses are ribonucleoproteins, the main protein being the nucleocapsid (N) protein (see Appendix A, Profile 27). Rice plants transformed with, and expressing, the N protein of RSV are protected against the virus (Hayakawa et al., 1992). iv. Mechanism of TMV CP Protection (reviewed by Prins et  al., 2008)  In the early experiments on CP-mediated protection the role of the plant’s innate RNA defense system, RNA silencing, was not fully recognized and some of the reported protection may have not have involved the CP itself. However, there is good evidence that at least some of the protections involved the CP, and Koo et  al. (2004) noted that several lines of evidence suggested that the protection against TMV results from interactions between the transgenic CP and the CP of the challenging virus including: (i) transgenic plants expressing CP show high protection to challenge by virions, but not to inoculation with RNA or partially stripped virions (Powell-Abel et  al., 1986; Register and Beachy, 1988); (ii) transgenic plants expressing TMV CP show greater levels of protection against closely related viruses than to more distantly related viruses (Nejidat and Beachy, 1990); (iii) transgenic plants expressing mutant CPs affecting electrostatic interactions between the subunits show modified protection according to their self-assembly capacity (Bendahmane et al., 1997). The molecular mechanisms of CP-mediated protection are not fully understood, and appear to be different for different viruses (Bendahmane et  al., 2007). Several hypotheses have been proposed for the mechanism of TMV CP protection. One mechanism suggested for protection against TMV is that it is associated with the certain configurations of quaternary structures formed by the CP rather than by the subunit itself (Asurmendi et  al., 2007). These different quaternary structures could be influenced by differences in the sites of carboxyl-carboxylate interactions between subunits (see Chapter  3, Section II, B, 1, e) (Bendahmane and Beachy, 1999). It is suggested that the degree of regulation of replication by CP

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aggregates determines the relative strength of the protection (Bendahmane et al., 2007); the regulation of replication could possibly be due to the CP aggregates affecting the balance between uncoating and coating incoming virus particles (Register and Nelson, 1992; Bendahmane et al., 1997). A second hypothesis proposed that a cellular site for TMV disassembly is blocked by the transgenic CP. To examine this hypothesis Clark et al. (1995) postulated that if the recognition site for binding transgenic CP to a putative receptor site is on the radial surface, the level of resistance against a challenging virus containing SHMV sequences on its surface would be similar to the resistance against SHMV itself. However, plants expressing TMV CP were just as resistant to the chimeric virus as they were to TMV. Plants challenged with a TMV mutant in which the CP was replaced by that of SHMV showed the same low level of resistance as that to SHMV. This experiment gave a strong indication that the binding site hypothesis was not tenable. However, the fact that protection is induced by viruses that have different forms of CP interaction in forming particles and that there are different levels of protection suggests that there are several mechanisms involving the CP itself. Furthermore, protection might be initiated by a combination of the CP itself and by RNA silencing induced by the CP transcript. An example of a combination of both mechanisms is shown in transgenic N. benthamiana containing the TCV CP gene (Vasudevan et al., 2008). b.  Viral Movement Proteins (reviewed by Prins et al., 2008) The proteins encoded by viruses that facilitate their cellto-cell movement are described in Chapter 10, Section IV, B. The relationship between both structure and function of movement proteins (MPs) indicated that, if one could block the function by, say using a defective mutant protein, a broader resistance might result (Cooper et al., 1996). Infection is delayed in plants transgenic in dysfunctional TMV MP (P30) which acts as a dominant negative mutant (Lapidot et al., 1993; Malyshenko et al., 1993). A TMV dysfunctional MP transgene interferes with systemic spread of the tobravirus TRV, the caulimovirus PCSV, and the nepovirus TRSV, but not the cucumovirus CMV (Cooper et  al., 1995); on the other hand, the functional analog of the MP increases susceptibility. As described in Chapter  10, Section IV, B, 1, e, the movement of potex-, carla-, hordei-, and some furoviruses is mediated by three overlapping ORFs composing the triple gene block (TGB). Expression of a WCMV dysfunctional 13-kDa MP confers protection to the homologous virus, as well as to other TGB-containing viruses, but not to TMV (Beck et  al., 1994). Similarly, a PVX dysfunctional 12-kDa MP gives protection against PVX and other TGB viruses but not against PVY (Seppänen et al., 1997).

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The transgenic expression of the MPs of BDMV has a deleterious effect on the plant development (Hou et al., 2000). c.  Viral Replicase and Replication-Associated Proteins (reviewed by Prins et al., 2008) i. RNA-dependent RNA Polymerases Replicase and RdRP sequences from several RNA viruses expressing proteins have been described as sources inducing protection including: AMV (Brederode et  al., 1995), CMV (Palukaitis and Zaitlin, 1997; Azadi et  al., 2011), PEBV (MacFarlane and Davies, 1992), PVX (Braun and Hemenway, 1992), PVY (Audy et  al., 1994) and TMV (Golemboski et al., 1990). In all these cases, the insert was a mutated or potentially dysfunctional RdRP, and in most cases the cognate protein was detected suggesting that the insert might be acting as a dominant negative mutant. However, as with CP- and MP-induced protection it is not always clear that the replicase protein is acting on its own. Various observations discussed by Palukaitis and Zaitlin (1997) indicate that this form of protection might not be just a dominant negative mutant effect but point to complex and subtle mechanisms. It is suggested that the interactions between the transgenic replicase proteins and other virus-encoded proteins may affect the processes of replication and cell-to-cell movement at the wrong time in the infection cycle leading to the arrest of some stage of the replication process. Furthermore, in at least some cases, it is suggested that the protection might involve RNA silencing as well as a protein-mediated mechanism. For example, the full-length 54-kDa RdRp from PMMoV, appears dispensable for induction of protection and plants expressing 30% of the protein are equally protected suggesting a dual protein- and RNA-mediated mechanism (Tenllado et  al., 1995, 1996). Expression of nine distinct overlapping segments covering the full TMV 183-kDa RdRp generates protection against the virus at low levels, and protein expression is not required. However, a higher protection is conferred by segments covering the polymerase domain of the protein, acting as dominant-negative mutants (Goregaoker et al., 2000). It was suggested that an initial RNA-based mechanism conferred by any sequence derived from the TMV genome is responsible for low-level protection followed by a more active protein-mediated mechanism, possibly in conjunction with RNA-mediated mechanism. ii. Geminivirus Rep Proteins  As described in Chapter 7, Section VIII, D, the genomes of plant ssDNA viruses do not encode polymerases, their replication requiring interaction between a viral replication-associated protein (Rep) and host polymerases. Transformation of N. benthamiana and tomato with a truncated TYLCV-Sar Rep protein gene strongly inhibits

virus replication in protoplasts and induces protection when expressed at high levels (Noris et al., 1996; Brunetti et  al., 1997). This dominant negative mutant, lacking a conserved NTP-binding domain, confers protection through two distinct molecular mechanisms depending on the challenging virus. In protecting against the homologous virus the transgenic protein inhibits the expression of the viral Rep protein; when transgenic plants are challenged with the heterologous TYLCV the transgenic protein forms dysfunctional complexes with the viral Rep protein (Lucioli et  al., 2003). However, the resistance is in some cases unstable due to transgene silencing. In contrast, tomato plants transgenic with a similar construct from a mild strain of TYLCV are protected against the homologous virus but are susceptible to a severe strain of the virus (Antignus et al., 2004). Similar strategies with Rep proteins mutated in the orior NTP-binding sites gave plants protected against other geminiviruses, such as BGMV (Hanson and Maxwell, 1999) or ACMV (Sangaré et al., 1997). Silencing the Rep gene of a nanovirus has also proved to give resistance against the cognate virus. Transgenic expression of an introns-hairpin-RNA construct the babuvirus BBTV Rep gene in banana plants confers a high level of resistance to virus infection (Shenhawat et al., 2012).

2.  Nucleic Acid-Based Protection (reviewed by Simón-Mateo and García, 2011) Four potential forms of protection based on the expression of viral RNA sequences have been recognized: (i) that induced by the viral RNA sequence expressed in (+) sense; (ii) that induced by the viral RNA expressed as antisense molecules; (iii) that induced by the expression of satellite RNAs; (iv) that in which ribozymes are targeted to viral genomes. Early in the development of CP-mediated protection, there were some unexpected observations especially in controls designed not to express protein. For instance, there was no correlation between the resistance and the expression of potyvirus, PVY, or luteovirus, PLRV CPs in potato (Kawchuk et al., 1990; Lawson et al., 1990). Lindbo and Dougherty (1992a,b) found that the untranslatable CP gene of TEV gave higher levels of protection than either full-length or truncated translatable constructs. Similar observations were made for protection given by untranslatable sequences of TSWV (de Haan et al., 1992) and PVY (van der Vlugt et al., 1992). The above and other observations suggested that the protection, at least in these cases, was mediated by nucleic acid rather than by protein. a.  RNA-Mediated Protection (reviewed by Prins et al., 2008; Runo et al., 2009; Collinge et al., 2010) As no promoterless transgenes have been shown to confer protection (Lomonossoff, 1995) it must be assumed

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that either the RNA transcript or a protein that is encoded give the protection. However, not all plant lines expressing a noncoding RNA give protection. As noted above, when a plant is transformed with a construct designed to produce a  viral protein it can often be difficult to distinguish between protection due to the expression of the protein itself or due to the RNA transcript. However, there are various features of the protection that tend to be characteristic for RNA-mediated protection (Smith et al., 1994): i. There is no direct correlation between RNA expression levels and the level of protection (Pang et al., 1993). ii. Usually, no transgene-encoded protein can be detected and the steady state of the transcript in inoculated plants is often in low amount. iii. The protection is usually narrow and against strains of the virus that have very similar sequences to that of the transgene. iv. Unlike CP-mediated protection, the protection is not overcome by inoculating RNA. v. Also, unlike CP-mediated protection, RNA-mediated protection is not dose dependent and operates at high levels of inoculum. vi. The insert in the host genome comprises multiple copies of the transgene, particularly with direct repeats of coding regions (Sijen et al., 1996). vii. Copies of the transgene may be truncated and/or in an antisense orientation (Waterhouse et  al., 1998; Kohli et al., 1999). viii. Transgene sequences and sometimes their promoter(s) may be methylated (Jones et  al., 1999; Kohli et  al., 1999; Sonoda et al., 1999). When transcript levels have been examined, three general classes of resistance phenotype have been recognized: i. Plants that are fully susceptible. These plants have low to moderate levels of transgene transcription and steady-state RNA. ii. Plants that become infected and then recover. These have moderate to high levels of transgene transcription and steady-state RNA in uninfected plants but low level steady-state RNA in recovered tissues. iii. Plants that are highly resistant have high levels of transgene expression but low steady-state levels. Figure 15.1 shows some of the features of RNAinduced protection. It is now generally accepted that RNA-mediated protection is involved in most cases of RNA-mediated protection. As described in Chapter 9 Section I, C, 1 dsRNA plays a major role in RNA silencing, and so the question arises as to how ssRNA constructs aimed at expressing proteins produce dsRNA. Usually, the existence of

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multiple copies of the transgene arranged in inverted repeats induce RNA silencing (Swaney et  al., 1995; Stam et  al., 1998); a single copy of the transgene may induce RNA silencing possibly by being transcribed to give dsRNA by host RNA-dependent RNA polymerase (Elmayan and Vaucheret, 1996; Dalmay et  al., 2000; Mourrain et al., 2000). Because of the variation in response, large numbers of independent transformants should be tested, not only to obtain lines with the best protection characteristics, but also to rule out the possibility that protection is not given by a particular construct. Since the recognition of the differences between proteinand RNA-mediated protection there have been many examples of plants transformed with viral sequences that show the properties of RNA-mediated protection. RNA silencing is now being increasingly used to provide protection against RNA viruses. Many of the constructs are now being based on hairpin-intron inserts (Figure 9.6). This approach is being used to give broadspectrum protection against several viruses in one crop, e.g. against PVX, PVY, and PLRV in potato (Arif et  al., 2012). b.  Transgenic Plants Expressing Antisense RNAs (reviewed by Tabler et al., 1998) One method of gene regulation in organisms is by complementary RNA molecules that are able to bind to the RNA transcripts of specific genes and thus prevent their translation. Such RNA has been called antisense or micRNA (messengerRNA-interfering complementary RNA). Appropriate antisense sequences incorporated into a plant genome have been shown to block the activity of specific genes (e.g., Delauney et  al., 1988; van der Krol et  al., 1988). Unintentional production of antisense RNA was considered to be a possible explanation of some of the anomalous results from attempts at CP-mediated protection and the possibility of using this strategy for the control of plant viruses has been explored. Various laboratories have carried out in vitro studies with oligonucleotides complementary to some plant virus RNA sequences. Oligodeoxynucleotides complementary to genomic PVX RNA causes translation arrest in a Krebs-2 cell-free system which is thought to be due to endogenous RNase H activity in the cell-free system (Miroshnichenko et  al., 1988). Antisense sequences complementary to sequences near the 5′ end of TMV RNA inhibit in vitro translation of this RNA in a rabbit reticulocyte lysate. The inhibition is probably due to direct interference with ribosome attachment (Crum et al., 1988). Morch et al. (1987) found that the “sense” nucleotide sequences corresponding to the replicase recognition site near the 3′ end of genomic TYMV RNA specifically inhibit in vitro the activity of the TYMV replicase isolated from virus-infected plants.

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Chapter | 15  Plant Viruses and Technology

Transgenic plant (sense / antisense / hpRNA / amplicon)

Nontransgenic plant

Efficient viral suppressor

Active transgene

Silenced transgene

dsRNA treated plant Mild viral suppressor

Heterologous viral infection Active transgene

Infection

Recovery

Resistance

Resistance

Silencing suppression

Silenced transgene

Recovery

Infection FIGURE 15.1  Schematic representation of natural and artificial RNA silencing-based antiviral resistance. Depending on the final outcome of the confrontation between defense/contra-defense mechanisms, different results of resistance, recovery, or susceptibility after virus infection can be obtained. From Simón-Mateo and García (2011) with permission of the authors.

The relevance of these various in vitro experiments to possible virus inhibition in vivo remains to be determined. Among transgenic tobacco plants containing genes for the production of antisense RNAs for three regions of the CMV genome, only one showed some resistance to the virus (Rezaian et  al., 1988). Cuozzo et  al. (1988) compared the extent of protection provided in transgenic tobacco plants by CMV CP gene or its antisense transcript. Symptom development and virus accumulation are reduced or absent in plants transgenic for the sense gene, and this is unaffected by inoculum concentration over the range used. By contrast, antisense plants are protected only at low inoculum concentrations. Transgenic tobacco plants expressing RNA sequences complementary to the CP gene of TMV are not protected as strongly from TMV infection as are plants expressing the CP gene itself (Powell et  al., 1989). In a few cases expression of antisense RNA gives significant protection, for example, to BYMV (Hammond and Kamo, 1995), albeit for only one season in a perennial

crop (Kamo et  al., 2005). Similarly, both sense and antisense constructs of TRSV CP give protection against the virus, apparently by an RNA-mediated mechanism (Yepes et al., 1996). Antisense RNAs also give protection against geminiviruses (reviewed by Shepherd et al., 2009). The targets have ranged from the rare mRNA of the Rep protein of TGMV and TYLCV (Day et  al., 1991; Bendahmane and Gronenborn, 1997; Yang et al., 2004) to the CP of ToMoV (e.g., Sinisterra et al., 1999). Antisense RNA has given some protection against viroids (reviewed by Tabler et al., 1998). c.  RNA-Mediated Resistance Against DNA Viruses Transformation of rice with a construct expressing RTBV ORF IV in both the sense and antisense orientation reduces replication of the cognate virus (Tyagi et al., 2008). These transgenes have been transferred into high-yielding rice cultivars which showed only mild symptoms on infection

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with RTBV in contrast to the usual severe symptoms (Roy et al., 2012). Transformation of hosts with a range of geminivirus constructs give protection against the cognate virus (reviewed by Shepherd et al., 2009). The main targets are the begomoviruses ACMV, TYLCV, and BGMV. As noted above, expression of sense and antisense RNA in transgenic plants have been employed successfully against TGMV (Day et  al., 1991), TYLCSV (Bendahmane and Gronenborn, 1997), and TYLCV (Yang et al., 2004), confirming the suggestion that RNA silencing can be harnessed for antiviral defense (Lapidot and Friedmann, 2002). In attempts to improve transgenic resistance further, Pooggin et  al. (2003) obtained recovery from virus infection in a transient assay using IR constructs containing the common region of the begomovirus VMYMV. Plants were made resistant to TYLCV by targeting antiviral resistance in transgenic plants the CP gene with an inverted repeat construct (Zrachya et  al., 2007). Similarly, Noris et al. (2004) and Ribeiro et al. (2007) produced transgenic plants expressing siRNAs against TYLCSV and ToCMoV, respectively. However, these plants often showed significant delays in symptom development, particularly at low inoculum dosage. In sharp contrast to the situation with RNA viruses, completely immune lines were not often observed. This may suggest that viral mRNAs are targets of RNA silencing, and that the success of the strategy depends on the relevance of the targeted gene product in the systemic spread of the virus. d.  Molecular Basis of RNA-Mediated Protection The recovery phenomenon associated with low steady states of transgene RNA in recovered tissues and the low steady-state RNA levels in highly resistant plants, coupled with the narrow range of protection against viruses with homologous sequences to the transgene are all suggestive of homology-dependent or post-transcriptional gene silencing (PTGS). This is described in detail in Chapter 9, Section II and reviewed in detail by Simón-Mateo and García (2011). Most studies on RNA-silencing-mediated antiviral protection have focused on viruses with (+)-stranded RNA genomes; RNA-silencing-mediated protection is also effective against (−)-stranded RNA viruses such as tospoviruses (Prins and Goldbach, 1998). In many cases the protection appears to follow the exogenous pathway (described in Figure 9.5). Transgene-derived RNA silencing against ssDNA viruses such as geminiviruses involves not only PTGS but also transcriptional gene silencing (TGS) (the epigenetic endogenous pathway; Figure 9.5) (Buchmann et al., 2009; Rodríguez-Negrete et al., 2009; Zhang et al., 2011). Although there is evidence that viroids can avoid RNA silencing through their structure (see Chapter  9, Section II, H) transgenic tomato plants expressing a viroid hairpin

Plant Virology

transgene and accumulating high amounts of viroid-specific siRNAs, show resistance to the homologous viroid, indicates that viroid RNA can be the target of RISCmediated degradation (Schwind et al., 2009). e.  Sequences for RNA-Mediated Protection RNA-mediated protection has been given by a range of sequences from viral genomes. In many cases, it has resulted from attempts to transform plants with the viral genes described above. Although RNA silencing can be induced by transgenes containing homologous sequences as short as 23–60 nt (Thomas et al., 2001) it is found to be more efficient with sequences longer than 100 nt (Pang et al., 1997; Jan et al., 2000b). RNA-mediated virus resistance is usually proportional to the sequence similarity between the transgene and the target virus and is usually not effective if the transgene sequence differs from that of the virus by more than 10%. As viral sequences in a quasispecies population will vary (see Chapter 2, Section III, A for quasispecies) it is a good strategy to use important functional sequences which are conserved such as those involved with replication. Various strategies have been devised to optimize the similarity between the transgene and the target (discussed by SimónMateo and García, 2011). As discussed by Helliwell and Waterhouse (2003), constructs giving intron-spliced hairpin RNA as among the most efficient at inducing RNA silencing. f.  Protein- and RNA-Mediated Protection A single type of construct may confer protection by both protein- and RNA-mediated mechanisms. For example, transformation with the TSWV N gene sequence confers resistance to heterologous tospoviruses in plant lines with the highest levels of expressed protein, but the most effective protection to the homologous virus is in plant lines that had the lowest steady-state levels of RNA and little protein (Pang et al., 1993; Schwach et al., 2004). Similarly, when barley plants are transformed with constructs to express BYDV-PAV CP, some resistant lines have detectable levels of CP whereas others do not (McGrath et  al., 1997). From an analysis of protection by TMV replicase sequences, Goregaoker et  al. (2000) concluded that both RNA and protein sequences were involved in conjunction with the speed of the infecting challenging virus. By using low temperature or viruses with silencing suppressors Bazzini et al. (2006) showed that PTGS does not play a significant role in PVX CP-mediated protection. On the other hand, protection against APMoV by both translatable and non-translatable CP gene sequences is due to just gene silencing (Neves-Borges et al., 2001). Although it is likely that both mechanisms collaborate to protect plants against a range of viruses, it is also

Chapter | 15  Plant Viruses and Technology

possible that a weak RNA silencing, unable to confer complete viral resistance, can suppress the expression of the transgene and thus inactivate the protein-mediated resistance (Lucioli et al., 2008). Palukaitis and Zaitlin (1997) draw attention to the need to test large numbers of independent transformants, not only to obtain lines with the best protection characteristics, but also to rule out the possibility that protection is not given by a particular construct. g.  Conferring Protection by RNA Silencing of Host Genes There is an increasing number of host gene targets for RNA silencing-based protection some of which are described below. Nagy and Pogany (2010) discuss global genomic and proteomic approaches to identifying host factors as targets to induce resistance against tomato bushy stunt virus (TBSV). A large number of recessive resistance genes to plant viruses are eukaryotic translation initiation factors (eIFs) of the eIF4E or eIF4G family (Chapter  11, Section IV). The resistance is due to an incompatibility of the eIF to interact with the viral genome. Rodríguez-Hernández et al. (2012) showed that silencing cucumber-eIF4E (Cm-eIF4E) gene gives protection against several viruses (CVYV, MNSV, MWMV, and ZYMV). Silencing Cm-eIF4E appeared to have little effect on the plant itself and did not affect the expression of its isoform Cm-eIF(iso)4E. Overexpression of the wild potato eIF4E-1 variant Eva1 elicits PVY resistance in plants silenced for native eIF4E-1 (Duan et al., 2012). The expression of the RTBV promoter is regulated by the host transcription factors, RF2a and RF2b (Chapter 6, Section VIII, A). Overexpression of these two factors in rice mitigates the symptoms of RTBV and reduces virus accumulation but does not have significant impact on plant development (Dai et al., 2008; reviewed by Dai and Beachy 2009). CMV Y-satellite (Y-sat) produces yellowing symptoms in N. tabacum (see Chapter 5, Section II, B, 2, a for satellites) by producing siRNA-directed silencing of the chlorophyll biosynthetic gene, CHL1 (Smith et al., 2011). The symptoms of Y-sat infection are completely prevented by transforming tobacco with a silencing resistant variant of the CHL1 gene. h.  Antiviral Resistance Mediated by Artificial miRNAs In Chapter  9, Section V, A, 1, I describe how the RNA silencing induced by virus infection can interact with the miRNA pathway. A new RNA silencing technique has been developed by modifying an miRNA primary transcript to give an artificial miRNA (amiRNA) (Vaucheret et al., 2004). The use of constructs containing an miRNA backbone targeted to degrade the invading viral RNA using

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the tasi silencing pathway are potential candidates for controlling plant viral diseases (Figure 15.2A). Figure 15.2B shows the steps in making an amiRNA construct. A typical construction of plant amiRNA expression vectors includes three major steps: amiRNA precursor sequence is amplified through three pairs of primers, inserted to an entry vector and then transferred into a binary destination vector through gateway-assisted cloning (Schwab et  al., 2006); Yan et  al. (2011) describe a modification which makes the cloning process more efficient. An important choice is the selection of the miRNA backbone. Pre-miRNA159a, pre-miRNA167b, and pre-miRNA171a have been shown to give efficient silencing (Ai et al., 2011). The choice of the target sequence can also be important. Not all amiRNA constructs based on PVY CP are equally effective in preventing virus infection (Jiang et  al., 2011). Analysis shows that targeting sequences of loose structure is the most effective. Targeting the silencing suppressor gives good protection against PVX and PVY (Ai et al., 2011). In both these cases, the level of protection was correlated to the expression level of the amiRNA backbone and to the degree of homology of the transgenic viral sequence to the target. In another approach, multiple amiRNAs targeting conserved motifs of the (−)RNA genome of WSMoV confer robust protection (Fahim et al., 2012; Kung et al., 2012). amiRNA-mediated protection has been shown to confer protection against an increasing number of RNA viruses from various genera in various crops: for example, TYMV and TuMV (Niu et al., 2006), CMV (Qu et al., 2007; Duan et al., 2008; Zhang et al., 2011), PVX and PVY (Ai et al., 2011; Jiang et al., 2011), WSMoV (Kung et al., 2012), and WSMV (Fahim et al., 2012). Simón-Mateo and García (2011) discuss the potential of amiRNAs and note some results indicating that it might work and some possible difficulties with its use. i. RNA Silencing-Mediated Resistance Without Transgenesis  The direct delivery of dsRNA derived from the tobamoviruses. PMMoV and TMV, the potyviruses, TEV, PVY, and SCMV, and the alfamovirus, AMV, by mechanical inoculation or by an Agrobacterium-mediated transient expression system, interfere with infection of the cognate virus in a sequence-specific manner (Tenllado and Díaz-Ruíz, 2001; Gan et  al., 2010; Yin et  al., 2010). This approach cannot cure already infected plants or confer permanent protection but can protect plants for at least 5 days (Gan et al., 2010); it can also protect against both families of viroids (Carbonell et al., 2008). ii. Relationship Between Natural Cross-Protection and Protection in Transgenic Plants The mechanism for transgenic protection against a virus infection, especially CP-mediated protection, has been compared with natural cross-protection or mild-strain protection (Chapter  14,

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Plant Virology

(A) Viral sequence amiRNA precursor

Plant virus

amiRNA duplex

RISC

Viral RNA cleavage Translation inhibition

(B)

FIGURE 15.2  Use of artificial miRNA for conferring resistance. Panel (A) Schematic representation of antiviral activity conferred by transgenic expression of an artificial miRNA in a plant. From Simón-Mateo and García (2011) with permission of the publishers. Panel (B) Structure of Arabidopsis miR319a precursor and strategy for artificial miRNA. Subpanel (a) Structure of pre-miR319a (176 bp) presented as a hairpin. Subpanel (b) Structure for artificial miRNA. Primers containing amiRNA or amiRNA* were used to replace miR319 and miR319* sequences in the pre-miR319a. The generated amiRcps targeted different regions of the CP sequence. From Jiang et al. (2011) with permission of the publishers.

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Section IV, A). There are several similarities that have been used to support the idea: (i) in both situations the degree of resistance depends on the inoculum concentration, with high concentrations reducing the observed resistance; (ii) both are effective against closely related strains of a virus, less against distantly related strains, and not at all against unrelated viruses; (iii) in some circumstances cross-protection can be substantially overcome when RNA is used as inoculum rather than whole virus (Sherwood and Fulton, 1982; Dodds et  al., 1985). Similarly, the resistance of transgenic plants expressing the CP of several viruses is substantially but not completely overcome when RNA is used as inoculum; (iv) in classic cross-protection experiments, no cross-protection was observed between two rather similar viruses, AMV and TSV. In experiments with transgenic plants expressing their CP, high resistance to infection was observed against the homologous virus and none against the heterologous virus (van Dun et al., 1988). On the other hand, there appear to be some differences between natural cross-protection and CP-induced protection. When cross-protection between related strains of a virus is incomplete, the local lesions produced may be much smaller than in control leaves. This indicates reduced movement and/or replication of the superinfecting strain. Local lesions that form in transgenic tobacco plants expressing the PVX CP are smaller than those of the controls (Hemenway et  al., 1988), in line with the result for PVX. However, the reduced number of local lesions that do form on transgenic plants infected with TMV become as large as controls, indicating no block in replication or local movement once infection is successful. It is quite possible that there are several mechanisms that give cross-protection. One of them is likely to involve the PTGS host defense system and thus, to resemble RNAmediated protection.

3. Ribozymes (reviewed by Tabler et al., 1998; Walter and Burker, 1998) As described in Box 5.2, ribozymes are catalytic RNAs that can cleave at specific sites in complementary target RNAs. Since the ribozyme has to be complementary to the target viral sequence, it has been considered to be an antisense RNA with extra properties. Incorporation of a ribozyme into an antisense RNA to TMV gives no significant advantage over the antisense RNA itself (de Feyter et al., 1996) but constructs of PPV that contain a hammerhead ribozyme give stronger protection than the ordinary antisense RNA construct (Liu et  al., 2000; Huttner et  al., 2001). Rice plants containing a ribozyme directed against RDV RNA 5 segment display high resistance or delayed and attenuated symptoms (Han et al., 2000).

4.  Transgenic Satellite-Mediated Protection The general nature of satellite RNAs is described in Chapter  5, Section II, B, and the ability of some satellite RNAs to attenuate the symptoms of the helper virus is discussed there. The use of transgenically-expressed CMV satellite RNAs is reviewed by Morroni et al. (2008). When transgenic tobacco plants containing DNA copies of a CMV satellite RNA are inoculated with a satellite-free CMV isolate, satellite replication occurs (Harrison et  al., 1987). At the same time, CMV replication is reduced and disease symptoms are greatly attenuated. In untransformed plants the CMV isolate causes mosaic disease and stunting. In the transformed plants, no mosaic appears, and plants grow almost as well as healthy ones. These differences persisted for 14 weeks, the longest period tested. Furthermore, the same result was obtained in plants raised from seed of the transformed plants. When transformed plants are inoculated with tomato aspermy virus (TAV), there is a similar attenuation of disease symptoms but without a marked decrease in TAV genome synthesis. Jacquemond et  al. (1988) showed that tobacco plants transgenic for a CMV satRNA are tolerant to infection by aphids, the main method of field transmission for CMV. Similar results were reported for another satellite-virus combination, satTRSV and TRSV (Gerlach et  al., 1987). Tobacco plants that expressed full-length satTRSV or its complementary sequence as RNA transcripts increase their synthesis of satTRSV RNA following inoculation with TRSV, but virus replication is reduced and disease symptoms are greatly ameliorated. This protection is maintained for the life of the plants. Two distinct mechanisms of resistance were found in N. benthamiana with full-length of sequences of a mild variant of satGRV inoculated with GRV plus severe satRNA (Taliansky et  al., 1998). In one set of transformed lines, there are high levels of transcript RNA and the replication of both severe sat-RNA and GRV genomic RNA is inhibited. In the second set of plants there are low levels of transcript RNA and replication of severe sat-RNA, but not that of GRV genomic RNA, is inhibited. It was concluded that in the first set of plants both GRV genomic and severe sat-RNA replication is downregulated by the mild sat-RNA and in the second there is homology-dependent gene silencing of the severe sat-RNA. Resistant plants are also produced using only the 5′ terminal one-third of the mild sat-RNA. The use of satellite RNAs in transgenic plants to protect against the effect of virus infection has both advantages and disadvantages. The protection afforded is not affected by the inoculum concentration, as it is with viral CP transformants. The losses that do occur in transgenic plants because of slight stunting will affect only the plants that become naturally infected in the field, whereas if all

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plants are deliberately infected with a mild CMV-satellite combination they will all suffer some loss (Chapter  14, Section IV, B). Furthermore, the resistance may be stronger in transgenic plants than in plants inoculated with the satellite. Inoculation is not needed each season, and the mutation frequency is lower. Nevertheless, these are distinct risks and limitations with the satellite control strategy. The satellite RNA could cause virulent disease in another crop species or could mutate to a form that enhances disease rather than causing attenuation (Chapter 14, Section IV, B). Another risk is the reservoir of virus available to vectors in the protected plants. Lastly, the satellite approach will be limited to those viruses for which satellite RNAs are known.

5.  DI Nucleic Acid-Mediated Protection Defective interfering (DI) nucleic acids are described in Chapter  7, Section IV, C. They are mutants of viral genomes that are incapable of autonomous replication but contain sequences that enable them to be replicated in the presence of the parent helper virus. In many cases, they are amplified at the expense of the parent virus, ameliorating the symptoms induced by that virus. When such nucleic acids are transgenically expressed, infection with the parent virus mobilizes and amplifies them. Transgenic expression of DI RNAs was shown to protect N. benthamiana plants against the apical necrosis and death usually caused by CymRSV (Kollár et  al., 1993). N. benthamiana plants transformed to express the TBSV DI RNA are protected against that virus and closely related tombusviruses (CNV and CIRV) but are susceptible to a distantly related tombus-like virus (CymRSV) and unrelated viruses (BDMV, PVX, and TMV) (Rubio et al., 1999). Transformation of N. benthamiana to express the DI DNA of ACMV interferes with the replication of both genomic components of that geminivirus (Frischmuth and Stanley, 1991; 1993). Serial transmission from the transgenic plants leads to increasing numbers of asymptomatic plants with undetectable levels of viral DNA. The protection afforded by the DI DNA is confined to closely related strains of the virus. Similarly, the accumulation of the Logan strain of BCTV is reduced in N. benthamiana plants transgenically expressing the DI DNA of that virus strain (Stenger, 1994); however, there was no effect on other beet curly top virus (BCTV) strains.

D.  Other Forms of Transgenic Protection Various forms of protection against viruses have been shown for a variety transgenes that are not derived from viruses themselves. Some of these are described in this section.

Plant Virology

1.  Transgenic Plants Expressing PR Proteins The PR host proteins are part of a non-specific host defense reaction and are involved in the phenomenon of local acquired resistance. Treatment of leaves with salicylic acid induces certain PR proteins and inhibits AMV replication in such leaves. Hooft van Huijsduijnen et  al. (1986) isolated and cloned the mRNAs for some of these proteins. In principle, it might be possible to provide protection against certain viruses by using “transgenic” plants in which PR protein genes are expressed constitutively under the control of a suitable promoter.

2.  Antisense to β-1,3-Glucanase The β-1,3-glucanases are proteins believed to be part of a constitutive and induced defense system of plants against fungal infection. Unexpectedly, plants deficient in these enzymes due to expression of an antisense RNA show markedly reduced lesion size and number in the local lesion response of N. tabacum Havana 425 to TMV and in N. sylvestris to TNV (Beffa et  al., 1996). The mutant plants also showed reduced severity and delay of symptoms of TMV in N. sylvestris.

3.  Transgenic Plants Expressing Virus-Specific Antibodies (reviewed by Stoger et al., 2005; Safarnejad et al., 2011) Plants do not have an immune system like that of animals in which specific antibody proteins are formed in response to an infection, and it has long been assumed that plants could not produce such proteins. However, the work of Hiatt et al. (1989) demonstrates that this is possible. They obtained cDNAs derived from mouse hybridoma mRNA, transformed tobacco leaf segments, and regenerated plants. Plants expressing single gamma (heavy) or kappa (light) chains were crossed to produce plants in which both chains were expressed simultaneously. A functional antibody made up over 1% of leaf proteins. The ability of plant-derived antibodies (plantibodies) to inhibit plant viruses was initially demonstrated using a scFv (see Chapter 13, Section III, A for antibody structures) against AMCV CP (Tavladoraki et al., 1993); infection incidence was reduced and symptom development delayed. Since then there have been several examples of protection given by transgenic expression of antibodies directed against both structural and nonstructural proteins (Table 15.2). A further suggestion is to express anti-idiotypic antibodies to important binding sites (Martin, 1998). Antiidiotypic antibodies are antibodies made against the antigen-interacting portion of an antibody and thus, have the same surface conformation CP, MP or replicase.

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TABLE 15.2  Recombinant Antibody-Mediated Protection Against Plant Virus Diseases Virus

Target Proteina

Transformed Species

rAB Formatb Cellular Localization

References

ACMV

CP

N. benthamiana

scFv

Cytosol

Tavladoraki et al., 1993

ArMV

CP

N. benthamiana

scFv

Cytosol

Nölke et al., 2009

BNYVV

CP

N. benthamiana

scFv

ER

Fecker et al., 1997

CMV

CP

N. benthamiana

scFv

Cytosol

Villani et al., 2005

CNV

RdRp

N. benthamiana

scFv

Cytosol, ER

Boonrod et al., 2004

CTV

P25, major CP

Citrus aurantifolia

scFv

Cytosol

Cervera et al., 2010

CYVV strain 300

CP

N. tabacum

scFv

Cytosol

Xiao et al., 2000

GFLV

CP

N. benthamiana

scFv

Cytosol

Nölke et al., 2009

PLRV

P1 protein

Solanum tuberosum

scFv

Cytosol

Nickel et al., 2008

PPV

Nib protein

N. benthamiana

scFv

Cytosol, ER, nucleus

Gil et al., 2011

PVY

CP, NIa protein

N. tabacum, S. tuberosum

scFv, VH

Apoplast, cytosol

Xiao et al., 2000, GargouriBouzid et al., 2006, Bouaziz et al., 2009

RCNMV

RdRp

N. benthamiana

scFv

Cytosol, ER

Boonrod et al., 2004

TBSV

RdRp

N. benthamiana

scFv

Cytosol, ER

Boonrod et al., 2004

TCV

RdRp

N. benthamiana

scFv

Cytosol, ER

Boonrod et al., 2004

TMV

CP

N. tabacum,

Full size IgG, Apoplast, Cytosol, scFv plasma membrane surface

Voss et al., 1995, Zimmermann et al., 1998, Schillberg et al., 2000, Bajrovic et al., 2001

TSWV

Nucleoprotein, MP N. benthamiana, N. tabacum

scFv

Cytosol

Prins et al., 2005, Zhang et al., 2008

TYLCV

Rep

scFv-GFP

Cytosol

Safarnejad et al., 2009

N. benthamiana

Modified from Safarnejad et al. (2011). a CP = coat protein; MP = movement protein; RdRp = RNA-dependent RNA polymerase. b IgG=; rAb = recombinant antibody ; scFv = single-chain variable fragment; VH = variable heavy chain.

The transgenic expression of plantibodies has potential for the control of plant viruses and also for determining functions of plant proteins (Figure 15.3) (de Jaeger et al., 2000), including those involved in disease determination.

4.  Transgenic Plants Expressing 2′-5′ Oligoadenylate Synthetase In mammalian systems, interferons are effective antiviral molecules. When one of the components of the virusinhibiting pathway, 2′-5′ oligoadenylate synthetase, was expressed in potato plants it gave protection against PVX (Truve et  al., 1993). The virus concentration in transgenic plants was lower than it was in plants expressing PVX CP.

5.  Transgenic Plants Expressing Ribosome Inactivating Proteins Ribosome inactivating proteins (RIPs) deglycosylate a specific base in the 28S rRNA and prevent binding of elongation factor 2. RIPs have been isolated from several plant species. Transgenic plants expressing the pokeweed (Phytolacca americana) RIP (termed pokeweed antiviral protein, PAP) showed protection against several viruses including PVX, PVY, PLRV, and CMV with little effect on plant growth (Lodge et al., 1993; Cao et al., 2011). Plants expressing a low level of a C-terminal deletion mutant of PAP are resistant to PVX (Tumer et  al., 1997). As the intact C-terminus is required for toxicity and depurination of tobacco ribosomes in vivo, it was concluded that the

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Plant Virology

1. Competitive inhibition

5. Prohibiting protein complex formation

3. Substrate or ligand binding

Active

Not active

Active

Active

Not active

Active

Not active

Active

Not active

Not active

6. Mistargeting Active 2. Allosteric inhibition Active Not active

4. Interference with protein folding

Active

Active

Not active

Not active

Not active

Enzyme Substrate

Antibody Receptor

Reaction product Ligand

Membrane

FIGURE 15.3  Potential mechanisms of antibody-mediated in vivo modulation of protein or signal molecule activity. From de Jaeger et al. (2000) with permission of the publishers.

antiviral activity of PAP can de dissociated from its toxicity. Another RIP from pokeweed, PAPII, is less toxic than PAP and plants expressing it are protected against TMV and PVX (Wang et al., 1998); a similar protein, PIP2 from P. insularis, exhibits antiviral activity against TMV (Song et al., 2000). The pokeweed antiviral protein and its applications are reviewed in Tumer et al. (1999). Other RIPs that show antiviral activity include BAP from Bougainvillea spectabilis (Balasaraswathi et al., 1998) which protected against TSWV, trichosanthin (Lam et  al., 1996) which gave protection against TuMV, and dianthin (Hong et al., 1996) giving protection against ACMV.

6.  Transgenic Plants Expressing Ribonuclease Gene pac-1 RNA viruses replicate via a complementary strand and thus, are thought to have a dsRNA stage in their infection cycle (Chapter 7, Section IV, B). To attack these replication intermediates the yeast-derived dsRNA-specific RNase gene, pac-1, was transformed into tobacco plants (Watanabe et al., 1995). Transformed plants show a decrease in lesion numbers when inoculated with TMV and a delay in symptom appearance when inoculated with CMV or PVY.

7.  Human Cystatin C As described in Chapter 6, Section IV, C, 2, a, poty­viruses express their genetic information as a polyprotein that is cleaved by virus-specific proteases to functional proteins. One of the viral enzymes, HC-Pro, is a papain-like cysteine protease (Box 16.1). Human cystatin C, an inhibitor of cysteine proteases, interferes with the autoprocessing of PPV polyprotein by HC-Pro and, unexpectedly, has an inhibitory effect on the NIa protease, a serine protease (García et  al., 1993). It was suggested that the transgenic expression of such a protease inhibitor might protect plants against viruses that involve polyprotein processing in their infection cycle.

8.  Transgenic Plants Controlling Insect Vectors and Transmission of Viruses (reviewed by Westwood and Stevens, 2010) There are two basic approaches to transgenic targeting transmission of viruses by insect vectors: making the plants toxic to the vector and interfering with the interactions involved in virus transmission. Some examples of these are:

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The bacterium Bacillus thuringiensis produces a polypeptide that is toxic to insects. Different strains of this bacterium produce toxic polypeptides with specificity for different insect groups (Tzotzos et  al., 2009). Vaeck et  al. (1987) produced transgenic plants expressing the toxin gene that are protected against insect attack. Among the virus vector groups of insects, only the Coleoptera appear to be affected by the toxin from some strains of the bacterium. In principle, it might be possible to produce transgenic plants protected against beetle vectors of some plant viruses. ASAL, a novel lectin from garlic (Allium sativum), is toxic to hemipteran pests when administered in an artificial diet. Expression in rice of ASAL under control of the phloem-specific Agrobacterium rolC and rice sucrose synthase-1 promoters significantly reduces the transmission of the rice tungro viruses (RTSV and RTBV) by the green leafhopper (Nephotettix virescens) (Saha et al., 2006). As described in Chapter  12, Section III, G, 1, the whitefly endosymbiont GroEL binds to viruses belonging to several genera. N. benthamiana plants expressing the whitefly GroEL gene show a high level of tolerance to TYLCV and CMV but not to GVA or TMV (Edelbaum et al., 2009).

E.  Discussion on Transgenic Protection 1.  Levels of Protection The reactions of various forms of transgenic protection give a great range of responses. These vary from delay in symptom production for just a few days to complete immunity. This raises the question of definitions of resistance and protection. Many transgenic plant responses do not fit the definitions outlined in Table 11.1. For instance, can the delay of symptom expression by a few days really be called resistance? Hull and Davies (1992) discuss this point and they suggest the following definitions: i. Resistance to a virus is a property of the plant that reduces virus multiplication and reduces or prevents virus spread within the plant and/or symptom expression. ii. Protection  is a property conferred to a plant that interferes with the virus infection cycle (the virus infection cycle includes transmission from an infected to a healthy plant and the full systemic infection of the healthy plant). Thus, most of the phenotypes described above should be classed as protection. Hull and Davies (1992) also go on to suggest that there should be categorization of field protection with the seven levels describing the behavior of the transgenic plant which would enable the reader or listener to understand the level of protection afforded by the “gene” being discussed. However, there are some problems in adopting such a system. First, the level of protection may vary between

TABLE 15.3  Reactions of Transgenic Squash Inoculated with SqMV Reactions of Plants Inoculated at: 1 Week

3 Weeks

5 Weeks

Line

n

S

rec R

n S

rec R

n

S

Control

8

8

0

0

7 7

0

0

5

5 0

0

SqMV-22

9

9

0

0

9 9

0

0

7

7 0

0

SqMV-3

9

4

5

0

9 6

2

1

6

2 2

2

SqMV-127

9

0

2

7

9 0

0

9

9

0 0

9

rec

R

From Jan et al. (2000c) with permission of the publishers. Transgenic R1 plants expressing SqMV CP genes were germinated, kept in a greenhouse for 10 days, and subsequently transplanted to a plastic house. The three upper leaves of each plant of plants were inoculated at 1, 3, or 5 weeks post-transplantation. Plants were observed for at least 60 days. The reactions were grouped in three phenotypes: susceptible (S), typical systemic symptoms were observed at 8–14 days after inoculation (DAI); recovery (rec), systemic symptoms were observed at 8–14 DAI, but no symptoms developed on new leaves at 20–40 DAI; resistant (R), plants remained symptom-free throughout the experiment. n, Number of plants inoculated. Note that the R1 transgenic plants were hemizygous for the transgene inserts.

siblings in a transgenic line. In many reports on protection, results are quoted as percent or number of plants showing (or not showing) symptoms. Second, the level of protection may vary according to conditions such as temperature (Nejidat and Beachy, 1989; Chellappan et al., 2005). Third, the level of protection may vary with the generation of progeny from the transformant. Thus, there are certain difficulties in categorizing levels of protection. For instance, the level of protection may vary according to the plant developmental stage (Table 15.3) (Jan et al., 2000c) or possibly due to environmental factors.

F.  Field Releases of Transgenic Plants (reviewed by Kaniewski and Thomas, 1993) There have been concerns expressed about the release and use of plants modified by genetic manipulation. This has led to plants produced by this means being treated in a different manner to those produced by conventional breeding techniques and being subject to specific regulatory structures. In most countries that have regulatory structures for the release of transgenic plants, there are two stages in the field releases of such plants (reviewed by Tzotzos et  al., 2009). In the first stage, the plants are released under contained conditions directed by the provisions of the country’s biosafety regulatory structure. This stage of release essentially has two purposes: (i) to address any potential

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(A) Plants showing symptoms (%)

100 80 60 40 20 0 0

(B)

10

20 30 40 62 76 Days post inoculation

100 Plants showing symptoms (%)

problems that a risk assessment might identify; (ii) to assess the field performance of the transgenic lines being tested. Plant lines that satisfy both the regulators and the releasers then go to the second stage of more general release, termed commercial release or farmer release. In this section, I will discuss field performance of transgenic plants and in the next section the possible risks of transgenes that protect plants against viruses. Testing the field performance of transgenic plants is essentially no different to testing plant lines that have been obtained by traditional breeding (Delannay et  al., 1989). The testing objectives include evaluating the plant appearance, typeness, growth vigor, yield, and quality. Of especial importance is to assess the stability and durability of the protecting transgene under these conditions. Three main factors can affect stability and durability: (i) instability of the transgene; (ii) possible climatic effects on the expression of the transgene; and (iii) the presence of protection-breaking strains or isolates of the virus that are present in the viral ecosystem but were not recognized in the initial glasshouse tests. There have been numerous contained field trials of virus-protected transgenic plants and lines of some crops such as tomatoes, potatoes, and papaya are in more general release (Perlak et  al., 1995; Thomas et  al., 1997; Gonsalves, 1998). Here I will give some examples of the results obtained. Field experiments with tomatoes suggest that transgenic expression of TMV CP may be commercially useful in this host (Figure 15.4). The transgenic plants were partially resistant to TMV and to strains L, 2, and 22 of ToMV. In the field, no more than 5% of transgenic plants showed systemic disease symptoms compared with 99% for the control plants. Lack of visual symptoms was correlated with an absence of ToMV. In inoculated control plants, fruit yields were depressed by 26–35%. There was no evidence that expression of the CP gene reduced plant growth or fruit yield compared with uninoculated non-transgenic plants (Nelson et al., 1988). In Australia, most lines of potato cvs Kennebec and Atlantic containing the PLRV CP gene showed no measurable differences in agronomic performance when compared with non-transgenic lines in the absence of PLRV (Graham et  al., 1995). However, under conditions where PLRV was prevalent, one transgenic Kennebec line gave ~30% increased tuber yield over its non-transgenic counterpart (Barker and Waterhouse, 1999). In the United States, some Russet Burbank potato lines containing the PLRV CP gene showed low levels of protection against primary spread of the virus but marked reductions in the secondary spread (Thomas et al., 1997). There was significantly lower infection rates and lower final SMV incidence values in two SMV CP-transformed soybean lines than in non-transgenic controls (Steinlage

Plant Virology

80 60 40 20 0 0

10 20 30 40 Days post inoculation

61

FIGURE 15.4  Development of systemic symptoms of TMV infection under field conditions in tomatoes transgenic for CP. ○, non-transformed plants; □ and , plants of two lines of transgenic tomatoes (A and B) expressing TMV CP. Plants were inoculated on terminal leaflets of three successive leaves with TMV strain U1 at 10 μg/ml, 8 days after planting out in the field. Observations were made on 48 plants of each line. From Nelson et al. (1988) with permission of the publishers.

et al., 2002) showing that there was less and slower spread of the nonpersistent aphid-transmitted virus in the protected lines. The expression of CMV mild satRNA in tomato confers field tolerance to CMV (Stommel et  al., 1998). Yie et al. (1995) describe the rapid production of homozygous tobacco lines protected against CMV by mild satRNA. Three of these lines were highly protected against CMV under field conditions. Transgenic lines that are protected against a mechanically inoculated target virus may not be protected when the virus is inoculated by its natural vectors. For example, one line of potatoes expressing the PVY CP gave promising results on mechanical inoculation but was not protected against aphid inoculation (Lawson et al., 1990). Similarly, transgenic plants expressing TRV CP were not protected against nematode transmission (Ploeg et al., 1993). Of particular concern is the stability of the protection conferred by the transgene. There are several ways

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in which the protection can be overcome or break down including: i. There are many factors that may affect the stability of a transgene, including rearrangement during meiosis and methylation. These usually show up in early generations of a transgenic line and would be unlikely to occur in later generations that were released to the field. ii. Environmental conditions may affect the expression of a transgene. iii. Protection may not always be effective with different strains of a virus. For example, tobacco plants transformed to express the CP of strain PLB or TCM of TRV are resistant only to the strain whose CP is being expressed (Angenent et al., 1990). On the other hand, Nejidat and Beachy (1990) found that transgenic tobacco plants expressing TMV CP gene are resistant to ToMV and TMGMV but not to RMV. The protective effect against TMV in transgenic plants expressing CP is greatly reduced when tobacco plants are held at 35°C instead of 25°C. However, plants held in a 25°C/35°C night/day cycle retain resistance (Nejidat and Beachy, 1989). iv. As described in Chapter  9, Section IV, most viruses encode gene products that suppress RNA silencing. There is concern that the silencing suppression of one virus may overcome the transgenic protection against another. However, Wang et  al. (2001) reported that coinfection by CYDV did not compromise the transgenic protection against BYDV-PAV. v. The CaMV 35S promoter is widely used in constructs to express virus-protecting transgenes. However, in oilseed rape plants in which this promoter is being used to express transgenes it can be silenced on infection with CaMV (Covey et  al., 1997; Al-Kaff et  al., 1998, 2000).

G.  Potential Risks Associated with Field Release of Virus Transgenic Plants (reviewed by Hull, 1990; Hammond et al., 1999; Tepfer, 2002; Fuchs and Gonsalves, 2007; Tzotzos et al., 2009; Thompson and Tepfer, 2010) A major consideration for the use of plant lines transgenically protected against viruses is the potential risks that might arise from their release to the general environment. This has been discussed in many places (see reviews cited above). Essentially, there are three areas of potential risk, risk to humans and other animals, risks to the environment, and commercial risks (discussed in detail in Tzotzos et  al., 2009). Risks to humans and other animals are basically any potential deleterious effects that the transgenic product may have to food and animal feed.

Since virus-infected plants have been consumed for long periods of time it is considered to be unlikely that protection mediated by viral sequences would pose significant risks (see Hull et al., 2000 for an example of the discussion on this point). There are potential risks from other transgenes used for protection against viruses, for example, RIPs, and also from the transformation process. For many regulatory authorities this requires the assessment of the substantial equivalence of the transgenic crop compared to the unmodified crop. This often necessitates a range of tests as outlined in Tzotzos et  al. (2009) and examples of such an assessment relating to food safety of papaya protected against PRSV and to antiviral antibodies are given by Yen et al. (2011) and Di Carli et al. (2009), respectively. The potential environmental and commercial risks involve any possible effects of spread of the transgene to other crops and wild species, non-target effects, the agronomic properties of the transgenic line, and the durability of the protection. These considerations are not limited to viral transgenes but apply to most, if not all transgenes (Tzotzos et al., 2009). One specific potential risk for plants that have resistance or protection against viruses (and other pathogen) is the spread of the gene(s) into wild species thus affecting the influence that pathogen infection would have on the ecosystem. In this scenario, transgenic resistance genes would be no different to natural resistance genes and thus would resemble the basic comparator in risk assessment. The area of virus-protecting transgenes that has attracted specific interest is the use of viral sequences. The basic question that is asked is: What is the risk of any interaction that might arise between a virus or virus-related sequence integrated in the host genome and another virus superinfecting that plant? Three scenarios are considered: heteroencapsidation, recombination, and synergism.

1. Heteroencapsidation Heteroencapsidation involves the superinfection of a plant expressing the CP of virus A by the unrelated virus B, the expression of the virus A CP not protecting the plant against virus B. The risk is that the CP of virus A might encapsidate the genome of virus B thereby conferring on it other properties such as different transmission characteristics. Heteroencapsidation by transgenically expressed CP has been reported for closely related viruses, for example, CMV and AMV (Candelier-Harvey and Hull, 1993) and between potyviruses (Lecoq et al., 1993). In a broader study using unrelated viruses, the particles of which have different forms of stabilization, Candelier-Harvey and Hull (unpublished data) showed heteroencapsidation only occurs between closely related viruses, the particles of which have similar forms of stabilization.

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2. Recombination The concern here is that recombination between the transgene and superinfecting virus might lead to a new virus. Recombination involving integrated sequences is discussed in Chapter  7, Section IX, B, 3. A hybrid virus, made in vitro by replacing the 2b gene of CMV by its homologue from TAV, is significantly more virulent than either of its parents (Ding et  al., 1996). Recombination between two strains of CMV or CMV and TAV occurred at similar rates in plants coinfected with the pairs of viruses and those superinfected by one virus into plants transgenic in sequence of the other of the pair of viruses (Turturo et  al., 2008). Transgenic plums expressing PPV CP do not promote the emergence of new PPV variants by recombination under field conditions for up to 8 years (Zagrai et al., 2011). This potential risk is particularly pertinent to luteoviruses (Miller et al., 1997) and is discussed in detail by Aaziz and Tepfer (1999) and Thompson and Tepfer (2010). The interpretation of experiments to throw light on risks of recombination in transgenic plants must be taken with caution. First, selection pressures will only favor recombinants which are better suited to the host or environment than the superinfecting virus. Second, because of the positioning of the transgene in the chromosome and transcription of it in the cytoplasm there might be different compartmentalization to that in natural joint infections.

3. Synergism As described in Chapter 9, Section V, I, synergistic interaction between two unrelated viruses are potentiated by distinct virus sequences. Thus, there is a possibility that the effect of a superinfecting virus could be exacerbated by a transgene expressing a synergism-inducing sequence.

4.  Avoiding Risk Understanding the molecular interactions involved in the potential risk situations can lead to methods for the “sanitizing” of the transgene to avoid that risk. For example, as described in Chapter  12, Section III, A, 8, b, aphid transmission of potyviruses involves an amino acid triplet (asp, ala, gly; DAG) in the CP. Mutation of this in a PPV CP transgene abolishes the aphid transmissibility but does not affect the protection offered by the transgene (Jacquet et al., 1998). Similarly, mutation in the PPV CP gene suppresses particle assembly, heterologous encapsidation and complementation in transgenic N. benthamiana but retains protection against ChiVMV and PVY (Varrelmann and Maiss, 2000). The understanding of the factors involved in recombination (Chapter  7, Section IX, B) will lead to

Plant Virology

transgene constructs that lessen the possibility of new molecules being formed between the transgene and a superinfecting virus. Similarly, sequences that potentiate synergism, such as the potyvirus HC-Pro or the cucumovirus 2b genes (see Chapter 9, Section V, I, 1), can be avoided. Another area in which risk has to be minimized is testing transgenic plants for resistance to quarantined viruses. Monticelli et al. (2012) describe a procedure for assessing transgenic plums for resistance to PPV. In all these risk assessments, it is important to compare the transgenic situation with the non-transgenic situation. Thus, there is the possibility of the above potential risks occurring in mixed infections between viruses. This is discussed in Hammond et al. (1999).

II.  DISCUSSION AND CONCLUSIONS ON TRANSGENIC PROTECTION As discussed in Chapter  4, Section II, it is impossible to give precise measures of crop losses due to viruses. For a given virus, losses may vary widely with season, crop, country, and locality. Nevertheless, there are sufficient data to show that continuing effort is needed to prevent losses from becoming more and more extensive. Essentially, once a plant becomes infected with a virus it is not feasible to try to cure it. The approaches of heat treatment and meristem culturing (Chapter 14, Section III, A, 3) are in practicality only applicable to high-value crops. Three kinds of situations are of particular importance: (i) annual crops of staple foods such as grains and sugar beet that are either grown on a large scale or are subsistence crops and that under certain seasonal conditions, may be subject to epidemics of viral disease; (ii) perennial crops, mainly fruit trees with a big investment in time and land, where spread of a virus disease, such as citrus tristeza or plum pox, may be particularly damaging; and (iii) high-value cash crops such as tobacco, tomato, cucurbits, peppers, and a number of ornamental plants that are subject to widespread virus infections. Possible control measures have been classified under three headings: (i) removal or avoidance of sources of infection; (ii) control or avoidance of vectors; and (iii) protecting the plant from systemic disease. In principle, by far the best method for control would be the development of cultivars that resist a particular virus on a permanent basis. Experience has shown that viruses continually mutate in the field with respect to both virulence and the range of crops or cultivars they can infect. The problem for most crop-virus situation is the lack of resistance genes in natural sexually compatible species; thus, transgenic protection offers a possible answer. Because of the variation of viruses, it has been generally considered

Chapter | 15  Plant Viruses and Technology

that breeding for protection or the development of transgenic plants is unlikely to give a permanent solution for any particular virus and crop. It has been suggested for conventional resistance to pests and diseases that one should not put too strong a selection pressure on a viral population or else a resistance breaking strain will arise. However, Hull (1994) has argued that as variation arises from the replication of a virus, the more that one can inhibit the replication the less chance there is that variants will arise that can overcome the protection. Furthermore, if transgenic protection is targeted at highly conserved sequences or motifs that are essential for replication it is highly unlikely that any variant would be able to compensate for the defect. However, as well as the science involved in improving agriculture, the use of transgene technology has raised many public issues such as the impact of agricultural practices on the environment, on the food that this industry provides, and the ethics of overcoming the species barrier. Thus, the application of this technology to improving the world’s food security will involve many more issues than just the science and its application.

III.  USES OF PLANT VIRUSES IN PLANT MOLECULAR BIOLOGY Various properties of viruses such as ease of manipulation, high-level amplification, site-specific recombination, strong infectivity, enhanced translation, and compact and repetitive morphological structure have enabled their broad application, from basic research to product development, including the generation of robust expression systems. There is the potential for infective viral genomes to be used as vectors to express heterologous proteins and for control sequences from viruses to be used in other vectors. Furthermore, the well-defined structure of viral capsids enables peptide sequences to be expressed on the surface; the particles can then be used to induce antibodies against the introduced sequences. Viral capsids can also be used as scaffold for nanotechnology. The pharmaceutical and industrial uses of plant viruses will be described in Section IV and in this section, I will discuss plant viruses as vectors of heterologous proteins and sequences and some of the uses to which these can be put.

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dsDNA genomes, because cloned DNA of the viruses was shown to be infectious. Interest later extended to the ssDNA geminiviruses, and then to RNA viruses when it became possible to reverse transcribe these into dsDNA which could produce infectious RNA transcripts. The main potential advantages of a plant virus as a gene vector were seen to be: i. The virus or infectious nucleic acid could be applied directly to leaves, thus avoiding the need to use transformation technologies and the consequent difficulties in plant regeneration; ii. The virus vector could replicate to high copy number; iii. There would be no “position effects” of insertion into a site in the plant chromosomal DNA; and iv. The virus vector could move throughout the plant, thus offering the potential to introduce a gene into an existing perennial crop such as orchard trees. Such a virus vector would have to be able to carry a non-viral gene (or genes) in a way that did not interfere with replication or movement of the genomic viral nucleic acid. Ideally, it would also have the following properties: (i) inability to spread from plant to plant in the field, providing a natural containment system; (ii) induction of very mild or no disease; (iii) a broad host range, which would allow one vector to be used for many species, but would be a potential disadvantage in terms of safety; and (iv) maintenance of continuous infection for the lifetime of the host plant. The major general limitations in the use of plant viruses as gene vectors are:

by Scholthof et al., 1996; Lico et al., 2008)

i. They are not inherited in the DNA of the host plant, and therefore genes introduced by viral vectors are expressed transiently and cannot be used in conventional breeding programs; ii. Plants of annual crops would have to be inoculated every season, unless there was a very high rate of seed transmission; iii. The foreign gene introduced with the viral genome may be lost quite rapidly by recombination or other means with the virus reverting to wild type; and iv. It would be necessary to use a virus that caused minimal disease in the crop cultivar. The viral vector might mutate to produce significant disease, or be transmitted to other crops that were susceptible. Infection in the field with an unrelated second virus might cause very severe disease.

In the early 1980s, there was considerable interest in the possibility of developing plant viruses as vectors for introducing “foreign” genes into plants. At first, interest centered on the caulimoviruses, the only plant viruses with

There are two basic forms of vectors, expression vectors in which the inserted gene is expressed as a protein or peptide and virus-induced gene silencing (VIGS) vectors in which the inserted sequence silences the expression of

A.  Plant Viruses as Gene Vectors (reviewed

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a targeted host gene. Lico et al. (2008) noted four types of viral expression vectors: i. Gene insertion vectors in which the foreign gene, usually accompanied by expression control sequences, is inserted into the viral genome. ii. Gene substitution vectors in which the foreign gene is inserted into the viral genome replacing one or more viral genes which are not essential for the replication and cell-to-cell movement of the recombinant genome. The foreign sequence is often expressed from a subgenomic RNA. iii. Modular or deconstructed vector systems which are based on dividing the viral genome into different elements (one or more of which contain the foreign sequence) which, when coinoculated, complement each other to establish a full infection. These modular elements are often cloned into Agrobacterium binary vectors and introduced into the plant by agroinfection or agroinfiltration. These vectors are reviewed by Gleba et al. (2005; 2007). iv. Peptide display vectors. These are discussed in Section IV.

1.  DNA Viruses as Gene Vectors a. Caulimoviruses Howell et  al. (1981) inserted an eight-base-pair EcoRI linker molecule into the large intergenic region of cloned CaMV DNA (see Appendix A, Profile 7 for CaMV genome organization) and showed there is no impairment of infectivity. Gronenborn et  al. (1981) demonstrated the successful propagation of foreign DNA in turnip plants using CaMV as a vector but they found that the size of the DNA insert that could be successfully propagated is limited to about 250 base pairs or less. Brisson et  al. (1984) reported the first successful expression of a foreign gene in plants using CaMV as a substitution vector. They used the 234-bp dihydrofolate reductase gene from E. coli, which confers methotrexate resistance in the bacterium. They inserted the gene into a derived strain of CaMV from which most of gene II (encoding the aphid-transmission factor) had been deleted. The chimeric DNA was stably propagated in turnip plants and the bacterial gene was expressed, as shown by assays for methotrexate resistance. de Zoeten et al. (1989) replaced the ORF II of CaMV DNA with the human interferon (IFNαD) gene. They obtained a stable strain of CaMV that replicated in Brassica rapa and led to the production of IFNαD. The interferon was located in the CaMV viroplasms and it had antiviral activity in an animal cell assay. Paszkowski et  al. (1986) constructed a hybrid CaMV genome containing the selectable marker gene neomycin

Plant Virology

phosphotransferase type II, which replaced the gene VI coding region. This construct was not viable in plants and could not be complemented in trans by wild-type CaMV. However, inoculation of Brassica campestris protoplasts under DNA uptake conditions gave rise to stable cell lines genetically transformed for the marker gene. This occurred only when the hybrid was coinoculated with wild-type CaMV. The mechanism for this effect is not understood. CaMV as a probe for studying gene expression in plants is discussed by Pfeiffer and Hohn (1989). Thus, there appear to be several constraints to the use of CaMV as a gene vector. These include the packing capacity of the CaMV particle, the amount of viral DNA that can be removed without affecting the functioning of the genome and the interactions between different parts of the genome in expression and replication. Removal of non-essential regions of the genome should enable about 1000 bp of sequence to be inserted (Fütterer et  al., 1990), but it is not certain as to whether all this sequence is really non-essential. b. Geminiviruses (reviewed by Mor et al., 2003) Much attention has been focused on the geminiviruses as potential gene vectors because of their DNA genomes and because the small size of the genomes makes them convenient for in vitro manipulations. Nevertheless, this small size may restrict the amount of viral DNA that can be deleted (Davies et al., 1987). However, this is counterbalanced by the fact that for some geminiviruses a viable CP and encapsidation are not necessary for successful inoculation by mechanical means, or for systemic movement through the plant (Gardiner et al., 1988). There are other potential difficulties (Davies et  al., 1987). Recombination can occur to give parental-type molecules. Many geminiviruses are restricted mainly to the phloem and associated cells. However, the wide host range of the geminiviruses (compared with the caulimoviruses) makes them of considerable interest. The fact that some members infect cereal crops would be particularly useful, except for the fact that they are not seed transmitted and are mechanically transmitted only with difficulty. In any event, inoculations on the scale needed for cereal crops would be impractical. Nevertheless, various experiments have shown that at least some geminiviruses can be used to introduce and express foreign genes in plants. As discussed in Chapter 7, Section VIII, irrespective of the monopartite or bipartite nature of geminiviruses, only their intergenic and the complementary strand ORFs are necessary for replication. Thus, a popular strategy used in developing geminiviral vector backbone is the replacement of the CP gene with reporter genes. Expression vectors have been made from several begomoviruses by replacing the CP gene with the gene

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of interest, for example, TGMV (Hayes et  al., 1988) or AbMV (Krenz et  al., 2010). Ward et  al. (1988) isolated a CP mutant of ACMV DNA1 in which 727 nucleotides in the CP gene were deleted. This mutant is non-infectious. However, when the CP ORF is replaced by the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the CP promoter, infectivity is restored, virus spreads through the plant, and the CAT gene is expressed. Nevertheless, systemic movement of infectious constructs with larger than normal DNAs in the CP position exerts a strong selection pressure favoring derivatives of normal size (Elmer and Rogers, 1990). A vector has been developed based on the monopartite begomovirus AYVV that can shuttle between E. coli and tobacco BY2 cells (Tamilselvi et al., 2004). Transient expression systems allow for the rapid screening of DNA constructs designed to study the activities of, for example, promoter sequences and RNA processing signals, in cells, and as a preliminary screen in the construction of transgenic plants. In principle, viral DNA might be altered to provide plasmid-type vectors for high copy number and rapid expression of modified or foreign genes. The work of Elmer et al. (1988) with a geminivirus progressed some distance toward the construction of such a plant plasmid. Deletion derivatives of TGMV DNA A defined the minimal DNA fragment capable of self-replication as about 1640 bp or 60% of the A sequence. Hanley-Bowdoin et  al. (1988) showed that following agroinoculation of petunia leaf disks with TGMV DNA A, the viral CP gene is transiently expressed, beginning about 2 days after agroinfection. Constructs were then made in which the CP ORF was replaced by the bacterial CAT or β-glucuronidase (GUS) genes, under the control of the CP promoter. Following agroinoculation, these foreign genes are also transiently expressed in petunia leaf disks. Gröning et  al. (1987) found that the DNA of AbMV infecting Abutilon sellovianum is located in the chloroplasts, raising the possibility of constructing a chloroplastspecific transformation vector. Although mastreviruses such as MSV can replicate without sequences such as that encoding the CP, they have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. Attempts to produce a complementing system to overcome these difficulties resulted in recombination between the two complementing viral constructs which would remove any inserted foreign sequences (Palmer and Rybicki, 2001). Similarly, a fully replication-competent, but truncated curtovirus, BCTV, viral vector lacked the ability to spread systemically (Golenberg et  al., 2009). However, this could be used to silence host genes (Section III, C) as the silencing signal would spread; thus replication but not spread of the vector is required for this function. This

vector could be useful for VIGS experiments as BCTV has a broad host range. Thus, the use of geminiviruses as vectors is often constrained by the inability to replace viral genes with “genes of interest” without compromising the replication of the vector, the propensity to recombination between the viral sequences, and the lack of integration of geminivirus DNA into the host genome. Various strategies have been developed to overcome some of these constraints. For example, using the dicot-infecting BeYDV, the Rep is either replaced by, or included with, the transgene. Replication of the expression vector is then driven via Rep or other viral proteins provided in trans or in cis, expressed from a cotransfected construct, from a Rep transgene stably integrated into the host genome (Mor et  al., 2003; Hefferon and Fan, 2004; Zhang and Mason, 2006; Regnard et al., 2010). An alternative to the use of the geminivirus genome as a vector is the use of a geminivirus satellite DNA (satDNA) (see Chapter 5, Section II, C for satellite DNAs). Li et al. (2007) showed that a heterologous DNA inserted in the TLCV satDNA is expressed in the presence of the helper virus; similarly TYLCCNV DNA β has been used as a vector (Cai et al., 2007).

2.  RNA Viruses (reviewed by Pogue et al., 2002; Bashir, 2007) The ability to manipulate RNA virus genomes by means of cloned cDNA intermediates has opened up the possibility of using RNA as well as DNA viruses as gene vectors. In principle the known high error rate in RNA replication (Figure 7.30) might place a limitation on the use of RNA viruses (Siegel, 1985; van Vloten-Doting et al., 1985). The experimental evidence to date suggests that mutation may not be a major limiting factor, at least in the short term. Viruses with rod-shaped and isometric particles have been studied as potential vectors, but those with rod-shaped particles have better potential as there are fewer constraints on the amount of nucleic acid that can be inserted. There are fewer limits to extending a rod-shaped particle to accommodate extra nucleic acid that there are to encapsidating the extra nucleic acid in a defined isometric particle. Some examples of the development of RNA virus vectors are given below: a. TMV Takamatsu et  al. (1987) prepared a TMV cDNA construct in which most of the CP gene was replaced by the CAT gene. When in vitro transcripts from this construct were inoculated onto tobacco leaves the local lesions that formed were smaller than normal, but biologically active CAT was produced, and this activity increased in the inoculated leaves for 2 weeks. RNA was extracted from these leaves and reinoculated onto tobacco after being

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encapsulated in TMV protein in vitro. CAT activity was again detected, indicating that this replicating RNA had some degree of stability in vivo. Yamaya et  al. (1988), using a disarmed Ti plasmid vector, introduced a cDNA copy of the TMV genome under the control of the CaMV 35S promoter into the genomic DNA of tobacco plants. This experiment demonstrates that a non-seed-transmitted RNA virus can be made seed transmissible. Dawson et al. (1989) constructed a hybrid TMV in which the CAT gene was inserted between the CP and the 30-kDa protein genes (Figure 15.5A). This construct replicates efficiently, produces an additional subgenomic RNA and CAT activity, and assembles into 350-nm virus rods. However, during systemic infection the insert is precisely deleted, giving rise to wild-type virus; it was thought that this deletion resulted from homologous recombination between the two copies of the CP gene subgenomic promoter. Takamatsu et al. (1990) constructed, via cDNA, a TMV RNA in which an additional sequence encoding Leu-enkephalin, a pentapeptide with opiate-like activity, was incorporated just 5′ to the termination codon for CP. The pentapeptide is expressed in protoplasts as a fusion product with CP. To overcome the problem of loss of the insert through recombination between homologous sequences, Donson et  al. (1991) developed a hybrid vector containing sequences from two tobamoviruses (TMV-U1 and ORSV (Figure 15.5B). In this vector, the gene of interest is expressed from the TMV CP subgenomic promoter and the CP from the ORSV promoter. There was a problem with partial deletion of one of the inserts, NPTII, but this was considered to be insert-specific. This vector system has been used to express α-trichosanthin (Kumagai et  al., 1993) and a derivative of this vector expressing the CP of ToMV instead of ORSV has been used to manipulate a biosynthetic pathway in planta (Kumagai et  al., 1995). Similar constructs have been made using a combination of homologous (TMV U1 CP) and heterologous (TMV U5 CP) subgenomic promoters (Roy et al., 2011). These basic vectors have been refined to optimize the production of recombinant proteins. Examples of the major factors that have driven this optimization are: i. High-level expression. Various approaches have been used to increase the level of transient expression of recombinant proteins from TMV vectors. A CaMV 35S promoter-driven vector lacking TMV CP sequence introduced by agroinfection gives high levels of expression and also improved biocontainment (Lindbo, 2007a). The expression level is also improved if such a vector is co-agroinfiltrated with a CaMV 35S promoter-driven gene for TBSV P19 silencing suppressor (Lindbo, 2007b). ii. Possibilities of recombination. As well as recombination leading to loss of the recombinant protein

Plant Virology

(A) CAT-CP 30K

CAT

CP

SPC

(a) CP-CAT 30K

CP

CAT

SPC

Hybrid TMV vector 30K

O-CP SPC

TMV-DHFR 30K

(b)

O-CP

DHFR SPC

TMV/NPTII 30K

NPTII

O-CP

SPC

(B)

U1-CP promoter

35s promoter

126/183 KDa

DsRed

MP DsRed 6 dpi

U5-CP promoter GFP GFP

pGRDualRed-GFP

Nos terminator

Area 1 (20X)

Area 2 (40X)

FIGURE 15.5  Panel (A) Schematic diagrams of TMV expression vectors. Subpanel (a) TMV mutants in which the gene coding for CAT is added into the complete TMV-U1 genome. The cat gene is fused behind the subgenomic promoter for the CP in order to produce an additional sgRNA. The resulting sequence duplications are indicated by arrows underneath each construct. Subpanel (b) Hybrid TMV expression vector in which all ORFs are TMV-U1 sequences, except the CP sequence, which originates from ORSV and is transcribed into mRNA from its own, ORSV-derived, subgenomic promoter (shown as a small open box). Foreign genes coding for DHFR and NPTII are expressed via subgenomic mRNAs transcribed from the heterologous TMV-U1 promoter located in the 30-kDa ORF. SPC, subgenomic promoter for the TMV-U1 CP. From Porta and Lomonossoff (1998) with permission of the publishers. Panel (B). Multigene expression from a chimeric TMV vector. Upper: Schematic diagram of a pGRDualRed-GFP construct. Lower: Microscopic observation of two different areas (with 20× and 40× magnification) of leaf tissue infiltrated with the pGRDualRed-GFP construct at 6 dpi. From Roy et al. (2011) with permission of the publishers.

discussed above, there is concern that there could be recombination with wild-type viruses on wide-scale commercial releases. This is discussed by Rabindran and Dawson (2001). Such problems of “contamination” could be reduced by using inducible viral vectors (Werner et al., 2011).

Chapter | 15  Plant Viruses and Technology

iii. Host range and movement within a host. The inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. DNA shuffling of the gene encoding the P30 MP improves the systemic movement of the recombinant vector (Toth et al., 2002). A vector based on SHMV gives expression in both legumes and Nicotiana (Liu and Kearney, 2010). iv. Expression of several proteins from the same vector. Coexpression of more than one protein from the same vector system has been achieved by using a combination of homologous and heterologous promoters or by developing a two-component vector system (Roy et al., 2010). v. Ease of manipulation. Lacorte et  al. (2010) describe PVX- and TMV-based vectors that are compatible with a commercial cloning system. However, some TMV-based vectors induce a hypersensitive-like response (HLR) in inoculated leaves of N. tabacum Samsun nn which blocks amplification of the recombinant protein (Li et al., 2010). The HLR is induced in susceptible tobacco through N/N’-gene independent pathways and is associated with the expression of the recombinant CP subunits. b. PVX A construct similar to that of TMV shown in Figure 15.5A was made with PVX with a duplication of the CP promoter (Chapman et  al., 1992). The PVX construct with the GUS gene being expressed from one of the promoters gives systemic infection of various Nicotiana species and retains the inserted gene especially in N. clevelandii plants. A PVXbased vector in which the TGB and CP were removed dramatically reduced the size of the vector genome (Komarova et  al., 2006). The addition of the gene for SPMV CP enhances the performance of a PVX vector (Everett et  al., 2010) through an unknown mechanism [SPMV CP is not a silencing suppressor (Qiu and Scholthof, 2004)]. Among some problems encountered in the use of the PVX vector is a negative correlation between the insert length possibly related to the duplicated subgenomic promoter and also to the inserted gene (Avesani et al., 2007). Also, a PVX vector contains a cryptic promoter which can affect plasmid construction in E. coli (Guo et al., 2007). c. TRV (reviewed by Ratcliff et al., 2001) Angenent et  al. (1989) found that sequences of 340 nts at the 5′ end and 405 nts at the 3′ end of the RNA2 of TRV (strain PLB) were sufficient for replication. Constructs in which the deleted viral nucleotides were replaced with a 1401-nucleotide sequence from plasmid DNA were replicated in protoplasts coinoculated with the complete genome of strain PLB, indicating that this virus may have

899

potential as a gene vector. A modified TRV-based vector which retains the transmission helper protein 2b gives efficient expression in roots (Valentine et al., 2004). TRV makes an effective VIGS vector (Section III, A, 3). d. GVA A vector based on the genome of GVA was constructed by duplicating the MP promoter using one derived from a distantly related isolate (Haviv et al., 2006). Duplication of the homologous MP promoter results in instability of the insert. This vector is useful for the improvement of grape vines and it also shows that vectors can be targeted to the desired crop by using an appropriate virus. e. CTV Several strategies have been examined for the development of vectors based on the complex RNA genome of the rod-shaped virus CTV which could be used in citrus trees (Folimov et al., 2007). Expression of a foreign gene is more stable by the gene-insertion strategy using either a duplicated CTV promoter or a BYV promoter than by the replacement strategy in p13 of CP ORF. f. Bromoviruses French et  al. (1986) constructed variants of BMV RNA3 in which the CP gene was replaced with the bacterial CAT gene, or in which the CAT gene was inserted near the 5′ end of the CP gene. When inoculated onto barley protoplasts together with normal BMV RNAs 1 and 2, these RNA3 constructs replicate and produce subgenomic RNAs equivalent to the normal CP subgenomic RNA (for subgenomic RNAs see Chapter  6, Section IV, C, 2, b). When the CAT gene is inserted in-frame with the upstream CP initiation codon, CAT expression exceeds that in plant cells transformed by Ti plasmid-based vectors. Pacha et  al. (1990) showed that all the cis-acting elements required for the replication of the 2.1-kb CCMV RNA3 can be contained in a 454-base replicon made of 5′- and 3′-terminal sequences. Thus, it may be possible to express a foreign gene of significant size using this replicon. g. TBSV TBSV CP is dispensable for systemic infection of certain Nicotiana species (Scholthof et  al., 1993) and hence can be replaced by foreign sequences such as the GUS gene (see Scholthof et al., 1993; Scholthof, 1999). By placing a cDNA copy of the TBSV genome between the CaMV 35S promoter and the hepatitis delta virus ribozyme followed by the nopaline synthase gene polyadenylation signal, Scholthof (1999) gained infections by directly rub-inoculating the construct. The CaMV 35S promoter and the NOS

900

terminator controlled transcription of the construct and the cleavage of the transcript by the ribozyme delimited the 3′ end of the infectious genome. This vector enables expression of inserts into the CP gene region. h. CPMV The genome of CPMV is divided between two isometric particles (Appendix A, Profile 29). The larger RNA-1 (5.9 kb) encodes a polyprotein that is processed to give products used in virus replication; the products of the processing of the polyprotein from the smaller RNA-2 (3.5 kb) form the viral capsid and facilitate cell-to-cell movement. Thus, additions to RNA-2 up to the size of RNA-1 should not affect encapsidation. To construct a CPMV vector, sequences of foreign genes were fused in frame with the polyproteins using the Foot-and-mouth disease virus 2A catalytic peptide to release the product; use of the CPMV protease cleavage sites resulted in loss of the insert through homologous recombination (reviewed by Liu et al., 2005).

3.  VIGS Vectors VIGS vectors differ from protein expression vectors in that they are directed at silencing the target gene. As described in Chapter  9, Section IV, most viruses encode genes that suppress RNA silencing which could also affect the silencing of the target genes. This presents a conundrum in that the viral vector requires silencing suppression for efficient replication but the silencing suppression could compromise the effect of the insert on the target. Obviously a balance between the two has to be achieved. TRV appears to be an effective VIGS vector (reviewed by MacFarlane, 2010) due, in part, to its very wide host range and induction of strong and fairly uniform silencing in tissues throughout the plant. Its VIGS efficiency is dependent on the plant species being used with N. benthamiana being the most responsive. The method of introducing the vector into the host can also influence the effects of the silencing suppressor. Hao et  al. (2011) found that using the Agrobacterium-mediated coinfiltration system (Brigneti et  al., 1998; Roth et  al., 2004) CNV p20 failed to act as a silencing suppressor although other experiments showed that it is a suppressor. See Section III, C for the use of VIGS vectors.

B.  Viruses as Sources of Control Elements for Transgenic Plants Certain plant viral nucleic acid sequences have been found to have useful activity in gene constructs as promoters of DNA and RNA transcription and as enhancers of mRNA translation.

Plant Virology

1. Promoters a.  Caulimovirus Promoters Transcription of CaMV DNA gives rise to a 19S and a 35S mRNA (Chapter  6, Section VIII, A) from separate promoters. Shewmaker et  al. (1985) introduced a full-length copy of CaMV DNA into the T DNA of the Agrobacterium Ti plasmid, and this was integrated into various plant genomes. They showed that the 19S and 35S promoters are functional in this form in several plant species. Both are strong constitutive promoters and have found wide application for the expression of a range of heterologous genes, the 35S being the promoter of choice in many systems. The 35S promoter also functions in cells other than those from plants (see Myhre et al., 2006). The 35S promoter is much more effective than the 19S in several systems. For example, expression of the α-subunit of β-conglycinin in petunia plants under control of the 35S promoter was 10–50 times greater than from the 19S promoter (Lawton et  al., 1987). The 35S promoter is 10–30 times more effective than the nopaline synthase promoter from Agrobacterium tumefaciens (García et al., 1987). Kay et  al. (1987) constructed a variant 35S promoter (the enhanced 35S promoter) that contained a tandem duplication of 250 bp of upstream sequences which give about a 10-fold increase in transcriptional activity. Various combinations of the cis-regulatory elements of the 35S promoter affect expression in different species (Benfey and Chua, 1990). The 35S promoter is nominally constitutive. However, Benfey and Chua (1989) showed that there is marked histological localization of expression of GUS activity in petunia under control of this promoter. By contrast, the 19S promoter directed expression of the CAT gene in a range of tissues in tobacco plants (Morris et al., 1988). The 35S promoter used in a selection marker gene can affect the expression pattern of a transgene (Yoo et  al., 2005). It is thought that the enhancer sequence within the promoter is responsible for the interference. Other viral promoters such as those from FMV (Maiti et  al., 1997; Bhattacharyya et  al., 2002), CsVMV (Verdaguer et al., 1996), MMV (Dey and Maiti (1999), and CERV (Holmberg et  al., 2002) have activity similar to, or in some cases even greater than, the 35S and 19S CaMV promoters in transgenic plants especially in specific tissues. For example, the FMV promoter facilitates stronger expression in soybean roots and nodules than do the CaMV 35S SVBV2 or CsVMV promoters (Govindarajulu et al., 2008). DNA shuffling of the FMV genomic and subgenomic promoters gives enhanced activities (Ranjan et al., 2012). dsRNAs can trigger methylation of the CaMV 35S promoter, which may be heritable, rendering it inactive and leading to transcriptional gene silencing (TGS); this phenomenon is especially notable in some hosts such as

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Gentian (Mishiba et al., 2010; Yamasaki et al., 2011). The length of the dsRNA above a threshold of 81–91 nt and the frequency of cytosines at symmetric sites in the CaMV 35S promoter are major factors allowing induction of this heritable TGS (Otagaki et al., 2011).

but their use in transgenic plants is limited because their expression often causes undesirable developmental effects. Silencing suppressors can be modified to be developmentally harmless (Saxena et al., 2011).

b.  Other DNA Virus Promoters Properties of promoters from other DNA viruses, such as badnaviruses and geminiviruses are discussed in Chapter  6, Sections VIII, A and B. Several of these promoters have been shown to have activity in transformation constructs especially those with specific properties such as tissue specificity. Some examples are: Among the badnaviruses, ScBV promoter is active in both monocots and dicots (Tzafrir et  al., 1998; Schenk et  al., 1999). This promoter is near constitutive in transgenic banana, N. benthamiana and Avena sativa; in Arabopsis it is constitutive in the rosette leaf stem, stamen, and roots and primarily limited to the vascular tissue of the sepal and silique. In these hosts the ScBV promoter has a similar activity level to the CaMV 35S promoter, but in Medicago sativa it has less activity that the CsVMV and CaMV 35S promoters (Samac et al., 2004). The TaBV promoter directs near-constitutive transcription but acts most strongly in the vascular tissues of banana and tobacco plants but directs less activity than the CaMV 35S promoter (Yang et al., 2003). The geminivirus WDV V-sense promoter (see Chapter  6, Section VIII, B for geminivirus promoters) drives phloem-specific expression in transgenic dicot species (Dinant et al., 2004). The MSV V-sense promoter shows an activation pattern restricted to the vascular tissues of aerial plant parts, in transgenic Arabidopsis, tobacco, and rice (Mazithulela et al., 2000; Escobar et al., 2010). Root expression is clearly detected in rice, but is absent in Arabidopsis and tobacco. The MSV promoter is highly active in nematode feeding sites at which giant cells are induced by three root-knot nematode species (Escobar et al., 2010). As the root expression is not induced by wounding it is suggested that the promoter activation during viral infection might share common features with nematode feeding site differentiation.

3.  Untranslated Regions as Enhancers of Translation

c.  RNA Viral Promoters The subgenomic promoters for several RNA viruses have now been defined (Chapter  7, Section IV, B, 1, c). These are used in the RNA virus vectors described above.

2.  Viral Silencing Suppressors The expression of foreign genes in plants is often hampered by the endogenous RNA silencing defense system. As described in Chapter  9, Section IV, plant viruses have silencing suppressor that can overcome this problem,

Untranslated leader sequences of several viruses act as very efficient enhancers of mRNA translational efficiency both in vitro and in vivo and in prokaryotic and eukaryotic systems (Chapter 6, Section IV, C, 3, e). AMV RNA4 is known to be a well-translated message for AMV CP. Jobling and Gehrke (1987) replaced the natural leader sequences of a barley and a human gene with AMV RNA4 leader sequence. These constructs show up to a 35-fold increase in mRNA translational efficiency in the rabbit reticulocyte and wheat germ systems. Sleat et  al. (1987) made similar constructs involving the uncapped mRNAs for two vertebrate genes and the bacterial GUS gene with or without a 5′-terminal 67-nucleotide sequence derived from the untranslated region of TMV RNA (the Ω sequence). The TMV leader sequence enhanced translation of almost every mRNA in the in vitro rabbit reticulocyte, wheat germ, and E. coli systems. Gallie et  al. (1987a,b) extended these results to show that the 67-nucleotide sequence was also a potentially useful enhancer in vivo in mesophyll protoplasts and Xenopus oocytes. A deletion derivative of Ω appears to be functionally equivalent to a Shine-Dalgarno sequence in several bacterial systems (Gallie and Kado, 1989). The translational enhancement brought about by the TMV Ω sequence is mediated by the ribosomal fraction of the in vitro system used (Gallie et al., 1988). The experiments of Sleat et  al. (1988) support the view that the untranslated viral leader sequences reduce RNA secondary structure, making the 5′ terminus more accessible to scanning by ribosomal subunits or by interaction with initiation factors. As described in Box 6.5, luteoviruses have translation enhancer elements. In a comparison of translation enhancer, the BYDV-like cap-independent translation enhancer (BTEs) was the most efficient of six tested for the expression of uncapped mRNAs in vitro and in vivo (Fan et al., 2012). The capped tobamovirus Ω sequence is the most efficient in tobacco cells. UTRs from SatTNV, TBSV, and crTMV do not stimulate translation efficiently; mRNA with the crTMV 5′ UTR is unstable in tobacco protoplasts.

4.  In Vitro Studies on Transcription In vitro translation systems which faithfully reproduce in vivo gene expression have proved very useful in

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animal and other systems for developing an understanding of nucleotide sequences and protein factors involved in the control of transcription. Cooke and Penon (1990) have taken a step in this direction for plant systems. They obtained a partially purified extract from tobacco cell suspensions that contained all the factors necessary for transcription from the CaMV 19S promoter.

5.  Use of the TMV Origin of Assembly for the Introduction of Foreign RNAs Sleat et al. (1986) showed that a correctly oriented origin of assembly (OAS) sequence located 3′ to a foreign RNA sequence initiates the efficient encapsulation of the foreign RNA in vitro. In an extension of these experiments, Gallie et  al. (1987c) prepared transcripts that encoded the OAS together with the RNA sequence for the CAT enzyme. When these were encapsulated in TMV CP in vitro and inoculated to a wide range of cell types the CAT mRNA was transiently expressed. Immunogold labeling located the site of disassembly and transient gene expression in epidermal cells of inoculated tobacco leaves (Plaskitt et al., 1987). Sleat et  al. (1988c) constructed a plasmid derivative containing the 5′ leader sequence of TMV followed by the CAT sequence and the OAS. This was introduced into the DNA of tobacco plants using an Agrobacterium vector. Transcripts from this nuclear DNA were encapsulated into TMV-Iike rods when the transgenic plants were infected with TMV. These experiments demonstrate an efficient complementation between functions encoded in the host genome and those of the infecting virus.

6. Ribozymes The potential of simple and specific RNA enzymes (ribozymes) from satellite RNA sequences is discussed above in Section I, C, 3.

C.  Viruses in Functional Genomics of Plants (reviewed by Godge et al., 2008; Purkayastha and Dasgupta, 2009; Becker and Lange, 2010; Senthil-Kumar and Mysore, 2011a) As plant genome sequence data accumulated, one of the main approaches to examining the functions of newly revealed genes involved reverse genetics by altering or knocking out that gene by transformation techniques and studying the mutant phenotype. This approach is laborious and time consuming, depended on knowing the sequence of the gene which is usually limited to model species such as Arabidopsis and rice and also on having a transformation system for the plant. The idea that viral

Plant Virology

systems could be used for functional analysis of host plants was conceptualized by Lindbo et  al. (1993) who suggested that, from experiments using plant transgenic with untranslatable transcripts of TEV CP, cytoplasmic regulation of gene expression may have important implications for a number of apparently unrelated plant phenomena. With the increase in understanding of gene silencing induced by virus infection of plants (Chapter 9, Section V) VIGS (Figure 9.2) was developed by expressing an antisense copy of the gene for phytoene desaturase in a tobamovirus vector in N. benthamiana; this turned off endogenous gene expression affecting the eukaryotic biosynthetic pathway (Kumagai et al., 1995). The term “VIGS” was first used to describe the phenomenon of recovery from virus infection (van Kammen, 1997) but is now widely used for the application of a viral vector to silence a plant gene. Since then VIGS has proved to be a potent tool for studying functional genomics of plants. VIGS involves three major steps: engineering viral genomes to include fragments of host gene(s) targeted to be silenced; infecting the appropriate plant host(s); and silencing the target genes as part of the defense mechanism of the plant against virus infection. There are numerous reports of the application of this technique, some of which are listed in the reviews mentioned above.

1.  Advantages and Disadvantages of VIGS Among the advantages of VIGS are: i. Avoids transformation. As noted above, transformation is time consuming and can be difficult with some species. VIGS can be used if there is a virus that infects that host and if that virus can be made into a VIGS vector. VIGS vectors have been made from DNA viruses as well as RNA viruses (Purkayastha et al., 2010). ii. Avoids problems with “lethal” genes. As the virus construct is inoculated to seedlings or mature plants it overcomes the problem with insertional mutagenesis (the other main approach to functional genomics) of identifying genes whose disruption is lethal before the plant has developed. With the VIGS approach, the lethality would be apparent from the death of the mature plant that had been inoculated. iii. Use at different growth phases of host. VIGS can be used to study genes that function at different stages in the growth of the host (Senthil-Kumar et al., 2008a). iv. Functional genomics of non-model species. As many plant genes have conserved regions in their sequence VIGS can be used on species, the genomes of which have not been sequenced to any extent. It is important that the silencing gene should have at least a 21 nucleotide sequence homology to the endogenous

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target gene (see below), but the full sequence of the gene does not necessarily need to be known. v. Rapid “fast forward” genetics. VIGS is rapid and so allows high-throughput screening. vi. Works in different genetic backgrounds. VIGS can be used on a range of varieties or cultivars of a host. vii. Tissue specificity. By choosing an appropriate promoter (Section III, B, 1) the expression of a gene can be suppressed in a specific tissue. viii. Ability to suppress multiple genes. By choosing appropriate insert sequences, VIGS can be used to silence multiple members of a family of genes (He et al., 2004) as well as a specific gene amongst closely related sequences (Senthil-Kumar et al., 2008a). Some of the disadvantages include: i. Lack of appropriate VIGS vectors. If there is not a VIGS vector for the host, a new one has to be developed. Some of the factors to be considered in developing VIGS vectors are discussed in Section III, B, 3. Examples of specific vector development are some proven and potential vectors for VIGS in grasses (Scofield and Nelson, 2009) and the development of BPMV-based vectors (Zhang and Ghabrial, 2006; Zhang et al., 2009). ii. Lack of an efficient method for delivering the VIGS vector. Although Agrobacterium binary vectors can be used from delivering VIGS vectors in many dicot and monocot species, some species are recalcitrant to this approach. In such cases, the VIGS virus can be multiplied in an agroinoculation-permissive host and then mechanically inoculated to the recalcitrant host. However, this approach will not work for viruses that cannot be mechanically transmitted though it may be possible to use their natural vector. iii. Silencing an unintended gene. As noted above, the silencing gene should have, at least, a 21 nucleotide sequence homology to the target. The selection of the silencing gene should be checked using publicly available software (Xu et  al., 2006; Senthil-Kumar and Mysore, 2011b) to find potential regions that generate efficient siRNAs for the target gene and have no sequence similarity with off-target genes. However, it must be recognized that the selection is only as goods as the database being used. iv. Uneven or localized VIGS. This may result from ineffective virus movement and the movement capabilities of the vector should be considered in developing the system. v. Virus symptoms and phenotypes. Virus symptoms can interfere with interpreting the silencing data and thus VIGS vectors that induce severe symptoms should be avoided. However, it is important to be aware that

silencing certain host genes can allow the virus to replicate to a higher level and thus cause more severe symptoms. If this is suspected, the virus titer should be assessed by local lesion or serological or PCR assays (Chapter  13). The virus vector can interfere with the plant metabolism and affect results from some plant– microbe interaction studies (Tufan et al., 2011). vi. Variation in silencing efficiency. This can be affected by insert length and orientation and the position of the target gene. For optimum VIGS the insert lengths should be ~200–300 bp (Burch-Smith et  al., 2006; Liu and Page, 2008) and should be checked using the publicly available software mentioned above. Of course, the orientation and position of the target gene cannot be changed, but this problem can be minimized by using a highly efficient vector. Gene silencing efficiency is not always correlated with percent homology of a heterologous gene sequence with the endogenous gene sequence; a 21 nt stretch of 100% identity between the heterologous and endogenous sequences is not an absolute requirement for gene silencing (Senthil-Kumar et al., 2008b). vii. Low silencing efficiency. VIGS seldom results in the complete suppression of expression of a target gene and, as a decreased transcript level could be sufficient to produce enough functional protein, an informative phenotype might not be observed in the silenced plant. Also, if the inserted sequence is not designed to target conserved gene sequences, phenotypes that are masked by functional redundancy between gene family members might be missed. viii. Nonuniform silencing. VIGS often does not result in uniform silencing of the gene throughout an infected plant. As noted in Chapter  10, Section V, E, 8 most viruses are excluded from meristematic tissues and thus, gene silencing in the meristem would not be possible. Furthermore, levels of silencing can vary between plants and experiments. This can complicate the interpretation of results, especially if the silencing does not produce a readily visible phenotype. Ways of minimizing these problems are discussed by Senthil-Kumar and Mysore (2011b). ix. Silencing suppressors. As described in Chapter  9, Section IV, most viruses encode genes that suppress RNA silencing; these would also affect the silencing of VIGS target genes and VIGS vectors have to take account of this.

2.  Future Potential of the Use of VIGS (reviewed by Senthil-Kumar, 2011b) As well as application to functional genomics there is an increasing number of potential uses of VIGS. For example,

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VIGS approaches, such as being combined with cDNAamplified fragment length polymorphism for confirming the identity of specific genes, can be used in molecular breeding programs and map-based cloning (Cakir et  al., 2010; Cheng et al., 2010). VIGS can also be used to produce new phenotypes of plants by down-regulating genes and thus altering metabolic pathways. This is especially useful in studies of phenotypes such as responses to abiotic and biotic stresses over a long period. Taken even further, if the phenotype persists over long periods it can be used to produce new traits. The potential for application in vegetatively propagated crops is shown by PVX-based VIGS in potatoes producing genotypically identical silenced plants on micropropagation (Faivre-Rampant et  al., 2004). There is an increasing number of examples of VIGS being transmitted through seed to the progeny of plants (Table 15.4).

IV.  USES OF PLANT VIRUSES IN INDUSTRY A.  Viral Vectors for Producing Recombinant Proteins (reviewed by Lico et al., 2008, 2012; Hefferon, 2012) There is increasing interest in the use of plant virus expression vectors as platforms for producing recombinant proteins which have a wide range of important applications, including industrial enzymes, new materials, biologically active peptides, vaccines and therapeutics for human and animal health, and components of novel nanoparticles for

various applications. Such platforms provide a system for cost-effective, highly scalable, and safe production of such recombinant proteins. Viral vectors (described in Section III, A) are proving useful in assembling and producing these recombinant proteins. Much of the production of industrial enzymes and new materials is by relatively straightforward expression of the gene of interest from the vector, and detailing these is beyond the scope of this book. However, the production of vaccines and nanoparticles has involved some “tweaks” of virus particle structure and are discussed below. As with transgenic protection against viruses, there are certain risks to humans and the environment that have to be considered in the industrial production of recombinant proteins. This is discussed by Fukuzawa et al. (2011) who describe a novel plant virus vector-helper plant system for the risk-managed production of bioactive recombinant proteins.

B.  Viral Vectors for Producing Vaccines and Pharmaceutical Proteins (reviewed by Yusibov et al., 2006; Lico et al., 2008; Rybicki, 2010) Several viruses, including the RNA viruses CPMV, TMV, PVX, CMV, AMV, and the geminiviruses BeYDV and BCTV have been engineered to produce vaccines, therapeutic proteins, and other proteins and peptides of potential pharmaceutical and industrial use (Yusibov et al., 2011). There are two approaches to using plant viruses to produce vaccines and other materials of medical and

TABLE 15.4  Potential VIGS Vectors Suitable for Long-Term Gene Silencing (from Senthil-Kumar and Mysore, 2011b) Virus

Plant Species

Seed Infection, Transmission and Stability of Virus

Potential for References Transmission of Silencing to Progeny

Seed Transmitted Viruses ALSV

Soybean

Embryo; transmitted through next generation

Low (20–30%)

Yamagishi and Yoshikawa (2011), Yamagishi and Yoshikawa (2009)

BSMV

Wheat

Embryo; transmitted through multiple generations

High (>80%)

Bruun-Rasmussen et al. (2007)

PEBV

Pea

Embryo and entire seed; uniform seed infection spread and silencing

Moderate (~50%)

Wang and Maule (1997)

TRV

N. benthamiana and tomato

Embryo and seed coat; transmitted through multiple generations

Low (10–15%)

Senthil-Kumar and Mysore (2011c)

Viruses Primarily Not Seed Transmitted CMV

Petunia and tomato Seed coat

Low (20–30%)

Kanazawa et al. (2011a), Kanazawa et al. (2011b)

PVX

N. benthamiana

Low (<10%)

Jones et al. (1999)

Seed coat

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veterinary interest, either expressing the target vaccine gene using a plant viral vector to transform plants or presenting the epitope on the CP of a plant virus. (Figure 15.6). As described in Chapter 13, Section III, A, 3, epitopes are patches of amino acids that adopt specific conformations. Free peptides can act as epitopes, but the immunogenicity is enhanced by presentation as multiple copies on the surface of a macromolecular assembly. One approach to presenting the peptide sequence in the correct conformation is to incorporate it into a viral CP sequence in such a way that it is exposed on the surface of the virus particle. The virus particle can then be used as a vaccine. There are several advantages to doing this with plant viruses including: (i) the virus can be produced in large amounts and in less-developed countries where the technology for animal virus vaccine production may be limited; (ii) vaccine proteins can be purified using a few simple steps (Paul and Ma, 2011); (iii) if the virus vector + transgene is integrated into the host genome plant/plant virus, systems can be transported and stored as seed; (iv) such vaccines may be given orally as part of the normal food supply inducing a strong mucosal response; (v) the virus will not infect the human or other animals and thus is completely inactive; (vi) the system is not subject to contamination by other virulent animal pathogens. A potential disadvantage is that the high rate of mutation of RNA viruses could result in the deletion or loss of inserted sequences, especially as they would not be under selection pressure (van Vloten-Doting et  al., 1985). Several plant viruses have been used for the presentation of foreign peptides including:

1. CPMV (reviewed by Liu et al., 2005; Sainsbury et al., 2010; Montague et al., 2012) The structure of CPMV has been solved to atomic resolution (Chapter  3, Section V, B, 6, a) (Lomonossoff and Johnson, 1991). The capsid comprises two types of protein, the L protein that has two β-barrel domains and the S protein that has one β-barrel domain. Analysis of the three-dimensional (3-D) structure suggested that loops between the β-strands would be suitable for the insertion of sequences to be expressed as epitopes as these loops are not involved in contacts between protein subunits. The βB-βC loop of the S protein is highly exposed (Lomonossoff and Johnson, 1995) and was used for most of the insertions (Figure 15.7); some insertions have been made in other loops (Lomonossoff and Hamilton, 1999). Early studies on inserting sequences at the βB-βC loop site gave guidelines for construction of viable, genetically stable chimeras (Porta et  al., 1994). These included (i) foreign sequences should be inserted as additions to, and not replacements of, the CPMV sequence; (ii) sequence

Gene encoding an antigenic protein from a pathogen.

Incorporate into a plant transformation vector for optimized expression in plant cells.

Stable expression: Chloroplast genome integration.

Stable expression: Nuclear genome integration.

Integrate into a viral coding sequence for expression as a “by product” of viral replication.

Transient expression: Infect plant to initiate viral replication.

Identify protective epitope within antigenic protein.

Modify viral genome to adapt it into a plant transformation vector for subsequent regulated release as a replicon in transgenic plants.

Create viral replicon coding sequence with epitope fusions to the virus coat

FIGURE 15.6  Plant-derived vaccine research strategies. From Arntzen et al. (2005) with permission of the publishers.

duplication should be avoided as this leads to loss of insert by recombination; (iii) the precise site of insertion is important for maximizing growth of chimeras. Understanding these guidelines gave a standard procedure for inserting foreign DNA into the βB-βC loop of the S protein (Spall et al., 1997). Initially, infection with CPMV-based chimeras was by inoculation with a mixture of RNA-1 and RNA-2-based transcripts generated in vitro by T7 RNA polymerase, but agroinfection was found to be easier and more efficient (Liu and Lomonossoff, 2002). The initial infections generally lead to only a relatively low yield of particles, but large quantities of modified virions are produced on passage to healthy plants. Generally, provided the inserted peptide is less than 40 amino acids long and has a pI below 9.0, the yields of modified particles obtained after infection with chimeras are similar to those obtained with wildtype CPMV (Porta et al., 2003). Expression of the foreign peptide did not alter the virus host range, increase the rate of transmission by beetles or through seed, or change the insect vector specificity. Although chimeric CPMV particles gave protective immunity in target animals (Sainsbury et al., 2010) there have been several problems, including reduced infectivity in plants, reduced or no yield on purification, and loss of all or part of the inserted sequence on viral passage; these have curtailed uptake of this technology for

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200K

VPg 32K Protease cofactor

An RNA 1

Q S Q M

Q S

58K Helicase

QG

4K 24K VPg protease

87K Replicase

105K95K VPg

AUG AUG 161 512

An QM

RNA 2

QG

48K Movement protein 58K

VP37 L

VP23 S

Coat proteins

Insertion point

COOH

βB-βC

S

A

B

L C

x 60

A

B C

S L

FIGURE 15.7  Generation of chimeric CPMV particles. Foreign sequences are inserted into the gene for the S CP borne by RNA2. Both RNA1 and RNA2 are translated into polyproteins and undergo a cascade of cleavages whose sites and final products are shown. RNA2 (bearing the heterologous sequence) needs to be coinoculated with RNA1 (unmodified) to initiate an infection in cowpea plants. S protein harboring a foreign epitope in its βB-βC loop and native L protein assemble at 60 copies each into icosahedral virus particles on which the foreign insert is expressed around the 5-fold axes of symmetry. From Porta and Lomonossoff (1998) with permission of the publishers.

Chapter | 15  Plant Viruses and Technology

907

FIGURE 15.8  Production of chimeric TMV particles in planta. Foreign oligonucleotide sequences are introduced at one of three positions, labeled A, B, and C, in the gene of the TMV CP, which is expressed from the most 3′ of the three viral subgenomic mRNAs. In vitro transcripts of the altered full-length genomic cDNA are inoculated onto tobacco. The resulting recombinant TMV CPs are represented as ribbon drawings with the numbers indicating each insertion site. Upon assembly of these CPs, chimeric rod-shaped virions are formed on which the foreign peptides are differentially displayed and distributed; a maximum of 5% of the CPs present an insert in position A. 100% of the CPs are modified in positions B and C. From Porta and Lomonossoff (1998) with permission of the publishers.

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Plant Virology

(A) Replicase

MP

CP

P35S

TNos

(B) Replicase

MP

RS

P35S

TNos X RS

GOI

CP TNos

P35S

FIGURE 15.9  Schematic representation of deconstructed version of Tobacco mosaic virus (TMV). (A) Full-length TMV expression vector. (B) Deconstructed expression vector for production of active replicons in plants. Virus replicase, MP, movement protein; CP, coat protein; RS, recombination site; P35S, CaMV 35S promoter; Tnos, nopaline synthetase terminator; X refers to the site of recombination. The constructs are delivered into plant cells via agroinfection; when a deconstructed virus is used, the two modules recombine at specific sites into the nucleus. From Hefferon (2012) with permission of the publisher.

commercial use. Montague et  al. (2011) discuss these problems and identify various ways by which they can be mitigated.

LIR

(A)

V1 Rep Intron

2. TMV (reviewed by Smith et al., 2009) With the development of infectious cDNA clones to TMV, it was possible to use a self-replicating system in plants. Fusion of a foreign sequence to the C-terminus of the CP prevents particle assembly (Takamatsu et  al., 1990). To overcome this problem, Hamamoto et al. (1993) placed the insert after an amber stop codon at the C-terminus of the CP gene (Figure 15.8A) so that it could be expressed as a readthrough protein. Particles were assembled with about 5% of the CP subunits expressing the inserted sequence (Sugiyama et  al. (1995). Replacement of two amino acids on a surface loop near the C-terminus of the CP gave particles with 100% of the subunits containing the insert (Figure 15.8B) (Turpen et al., 1995) as did insertion into another part of the C-terminal region not involved in particle assembly (Figure 15.8, C) (Fitchen et al., 1995). As discussed in Section III, A, 2, a, TMV can also be used as an expression vector by incorporating a separate subgenomic promoter into its genomic RNA. This approach has been used to express several proteins and peptides of pharmaceutical interest (Hefferon, 2012). Deconstructed TMV vectors in which the viral genome into two separate molecules, one containing the information for replication and the other containing cassettes for insertion of foreign genes (Figure 15.9) have enabled highlevel expression of biopharmaceutical proteins (Gleba et al., 2004; Santi et al., 2008; Noris et al., 2011).

MP

RepA C1

V2 CP

C2 SIR (B)

Rep 35S

Nos

Gene of interest 35S

Nos

FIGURE 15.10  Geminivirus expression vector constructed for expression of vaccine proteins. Subpanel (a) Genomic organization of Bean yellow mosaic virus. Subpanel (b) Deconstructed expression vector. LIR, long intergenic region; SIR, short intergenic region. From Hefferon (2012) with permission of the publishers.

3. Geminiviruses Geminiviral vectors have been used to express a range of proteins some of which are of pharmaceutical interest (Chen et  al., 2011). There are various forms of geminiviral vectors based on bipartite begomovirus genomes and on deconstructed monopartite genomes; see for example Figure 15.10.

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4.  Other Viruses Chimeric fusions to the CP of the potyvirus JGMV have been expressed in E. coli (Jagadish et al., 1993, 1996). The extracted protein could be polymerized into potyviruslike particles. These particles could be formed with inserts at the N-terminus or replacing some of the C-terminal sequence. A construct that gave both fused and unfused versions of the green fluorescence protein at the N-terminus of PVX CP is described by Oparka et  al. (1996). In this construct the GFP gene was separated from the CP by the Foot-and-mouth disease virus 2A sequence that mediates processing at its C-terminal end. The presence of some unfused CP molecules was essential for the assembly of virus particles. In subsequent experiments, proteins ranging in size from 8.5 to 31 kDa were expressed as “overcoats” on the surface of PVX particles (Santa Cruz et al., 1996). TBSV particles were assembled from the construct shown in Figure 15.11 in which an insert was made at the C-terminus of the CP gene (Scholthof et  al., 1996). Similarly, the Human immunodeficiency virus p24 ORF was expressed as an in-frame fusion with a 5′-terminal portion of the TBSV CP ORF (Zhang et al., 2000). Thus, it would appear that TBSV can tolerate insertions in both the N-terminal and C-terminal regions of the CP.

UAG readthrough 5'

33K

41K

92K

Polymerase

22K 19K

3'

41K

Coat protein

22K 19K Movement proteins

Insertion point

P COOH

S

(R) NH2

x 180

C.  Plant Viruses in Nanotechnology

(reviewed by Young et al., 2008; Lee et al., 2009; Aead, 2010) One of the basic features of nanotechnology is the selfassembly of nanometer-sized components to form devices. Biological macromolecular systems, such as virus particles, have considerable potential for forming the building blocks for nanotechnology for several reasons including: i. Virus particles are robust and can be produced in large quantities with relative ease. ii. Viruses self-assemble into monodisperse particles with a high degree of symmetry and polyvalency. iii. Virus particles can form ordered arrays. iv. As the basic units of virus particles (the CPs) are encoded by the viral genome, they are programmable by genetic engineering, and as they are proteins they can also be chemically engineered. Plant viruses are particularly attractive for use in nanotechnology as their production in plants can be easily scaled up and they are relatively easy to purify. Additionally they have simple genomes which are easy to manipulate and for biomedical uses they are not known to infect animals.

FIGURE 15.11  Generation of chimeric TBSV particles. Fusions of heterologous sequences are made at the 3′ end of the CP gene in a full-length cDNA clone of the viral genome. The CP is translated from the larger of the two viral subgenomic mRNAs and comprises three domains, designated R, S, and P, with P bearing the foreign amino acids (shown in black). When 180 copies of the viral CP, labeled A, assemble to form icosahedral particles, the fused peptides, each represented by a black half-circle, are well-exposed at the surface of the virions. From Porta and Lomonossoff (1998) with permission of the publishers.

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There are many potential uses for plant viruses in nanotechnology including: i. Modifying the surface structure of virus particles to produce reactive groups which are exactly positioned and which can serve as attachment sites for conjugation and precise display of heterologous materials (Figure 15.12). ii. Modifying the interfaces between viral subunits (Figure 15.12). iii. In vitro assembly of particles enabling different final structures and combination of new surface properties. iv. Modification of internal surfaces of virus particles to form vessels containing novel products (Figure 15.12). v. Formation of empty particles which can act as vessels which can also address biosafety and biomedical concerns. vi. The use of viral proteins which will degrade addresses medical and environmental concerns on nanotechnology based on non-degradable molecules (Warheit et al., 2008). The potential of an increasing number of plant viruses for use in nanotechnology is being explored (Young et  al., 2008). As many advances have been made with the isometric virus, CPMV, and the rod-shaped virus, TMV, I will describe some of approaches with these two viruses to illustrate the potential for the use of viruses in nanotechnology.

1.  Cowpea Mosaic Virus and Nanotechnology (reviewed by Steinmetz et al., 2009; Evans, 2010) CPMV has isometric particles with its genome divided between two species of ssRNA, RNA1 encoding a polyprotein processed to give products required for viral replication and RNA2 encoding a polyprotein giving the two capsid proteins, the small (S) and the large (L) subunits and the MP (for details of the genome organization, see Appendix A, Profile 29). There are 60 copies of each capsid protein arranged in a pseudo T = 3 symmetry (Chapter 3, Section V, B, 6, a). As the structure of CPMV particles is resolved to atomic level, the sequence of both RNA species is known, and there are infectious clones of both RNAs, various modifications have been made to the virus that enable use of both the outside and inside of the particles for nanotechnological applications. a.  Modifications for the Use of the Outside of CPMV Particles As described above (Section IV, B, 1), amino acid sequences can be added to surface loops (e.g., βB-βC loop in the S protein) without compromising virus assembly

FIGURE 15.12  Schematic representation of the three surfaces of plant viral capsid architectures, each of which can be modified to impart function by design. From Douglas and Young (2006) with permission of the publishers.

and structure. As there are no non-disulfide-linked residues on the surface of CPMV, sites with thiol-selective chemistry have been constructed either by inserting Cyscontaining peptides into surface loops or by mutation of appropriate residues on surface loops (e.g., βE-βF loop in the L protein) to Cys residues (Wang et  al., 2002; Blum et  al., 2005). In a similar approach, CPMV particles with controlled amine reactivity have been made by replacing surface lysine residues with arginine (Chatterji et  al., 2004a). CPMV can be crystallized giving 3-D arrays which contain large solvent channels. These repeating channels within the CPMV crystal have the potential to allow diffusion of nanomaterials into the crystal interior and binding to virus particles in an ordered manner structure. Thus the channels can be used for confined and regular growth of metals such as palladium and platinum (Falkner et  al., 2005). Cys-added CPMV mutants have been used to form a scaffold for bottom-up self-assembly forming conductive networks (Blum et  al., 2005). Similarly, networks have been constructed using modified virus particles together with carbon nanotubes and quantum dots forming hybrid structures (Portney et  al., 2005; 2009). Multilayered thin film assemblies have been achieved by layer-by-layer assembly of CPMV particles labeled with two different ligands on solid supports (Steinmetz et  al., 2006). Such structures have the potential for various uses in nanotechnology (reviewed by Steinmetz et al., 2009).

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The chemical conjugation of proteins to the outer surface of CPMV particles can enable modified particles to be targeted to cell surface receptors. For example, Herstatin-2 (Her-2) intron 8 is a receptor-binding module that binds to the epidermal growth factor receptor (EGFR) and Her-2 receptor. Chatterji et  al. (2004b) conjugated Her-2 intron 8 onto modified Lys and Cys CPMV CP to give modified particles that could be targeted to cells with EGFR and Her-2 receptors. (Figure 15.13). b.  Modifications for the Use of the Inside of CPMV Particles CPMV preparations comprise three types of particles separable by gradient centrifugations, bottom component containing RNA1, middle component containing RNA2 and a small proportion of top component containing no RNA (termed empty particles). These empty particles have considerable potential for loading and delivering various compounds, for example, biomedicals and drugs. As described in Chapter 6, Section V, G, the two CPMV capsid proteins, the L and S proteins, are expressed as part of the polyprotein encoded by RNA 2. Saunders et al. (2009) showed that coexpression of VP60 (the moiety of the RNA2-encoded polyprotein containing the L and S proteins) and the virus-encoded 24K proteinase in both insect cells and plants resulted in the release of the L and S proteins and formation of empty virus-like particles (eVLPs). These eVLPs can be loaded with metals and metal oxides by diffusion through pores in the particles (Aljabali et  al., 2010). The access to the interior surface of the eVLPs is controlled by the C-terminal 24 amino acids of the S protein and elimination of this peptide greatly increases the efficiency of mineralization (Sainsbury et  al., 2011). Replacement of this C-terminal peptide with six histidine residues also facilitates external mineralization with Co. Coupling the ability to modify CPMV particles to target cell receptors with the ability to form eVLPs that can be loaded with various materials opens many possibilities such as their use in drug delivery (see Figure 21 in Steinmetz et al., 2009).

2.  Other Isometric Viruses Approaches similar to those described above have been used for adapting RCNMV (reviewed by Lockney et  al., 2011) and CCMV particles (reviewed by Young et  al., 2008) for nanotechnology. Among the properties of these two viruses is the ability to change the interactions controlling the stability of the particles by adjusting pH and divalent cations (Chapter  3, Section I, J). This has led to suggestions for the possible use of these nanopaticles, for instance ones based on RCNMV, in cancer treatment (Franzen and Lommel, 2009).

FIGURE 15.13  CPMV for drug delivery. Therapeutics are loaded in the capsid interior and homing domains (Int 8) are attached for targeting of cancer cells. Once the virus particles are attached to the cells, they will be endocytosed, and the drugs will be released once the virus particles are degraded in the cytosol. From Steinmetz et al. (2009) with permission of the publishers.

3.  TMV and Nanotechnology TMV has several properties that make it attractive for use in nanotechnology. These include its high yield, high stability, the rod-shaped particles, end-to-end aggregation of particles to form rods longer than a single virion, knowledge of sequence and infectious clones of its RNA genome, knowledge of the CP structure at atomic resolution and the ability to self-assemble particles from separated CP and RNA, the length of particles being dependent on the length of RNA. The structure of TMV particles is described in Chapter 3, Section II, B, 1 and the assembly of particles in Chapter 3, Section III, A, 2. Different chemical groups of the CP can electrostatically, or as ligands, bind metal ions. For example, after activating particles by the selective binding of Pd(II) or Pt(II) ions, metallization with Ni or Co forms 3-nm “wires” within the central channel (Figure 15.14A) (Knez et  al., 2003). Replacement of the glutamic acid of a Caspar-carboxylate group (Chapter  3, Section II, B, 1, e)

(A)

(B) 100 nm

(a)

(b)

(C)

(a)

100 nm (c)

(D)

100 nm

100 nm

(a)

(b)

100 nm

(b)

100 nm (d)

400 nm

(E)

1 µm

(c)

FIGURE 15.14  Some nanotechnological applications of TMV. Panel (A) Wires. Subpanel (a) TEM image of TMV after Pd(II) activation, followed by electroless deposition of Ni. Two adjacent virion aggregates are filled with wires; the left aggregate comprises three TMV particles whose combined channels are filled to a length of 600 nm. Energy-resolved scanning TEM images proved that the dark wire is indeed composed of Ni. Inset (top left): A single virion is filled with a 200 nm-long wire with a ca. 3-nm diameter. Subpanel (b) TEM image of TMV after Pd(II) activation, followed by electroless deposition of Co. The virion is filled by a 200 nm-long wire with a ca. 3-nm diameter. From Knez et al. (2003) with permission of the publishers. Panel (B) Schematic setup of the field-effect transistors. From Atanasova et al. (2011) with permission of the publishers. Panel (C) Nanodumbells. TEM [subpanels (a), (b), and (d)] and, topographical images [subpanel (c)] of Au nanoparticles (red arrows) bound to TMV and TMV-RNA. Subpanel (a) A single TMV containing one Au nanoparticle at each end. Subpanel (b) Two virions aggregated end-to-end; the ends of the rod contain bound Au nanoparticles. Subpanel (c) A single TMV (ca. 13 nm high) on a silicon substrate containing Au nanoparticles (5–8 nm high) at each end. Subpanel (d) Au nanoparticles bound to coiled TMV-RNA; the inset shows an expanded picture (350 nm × 350 nm). From Balci et al. (2007) with permission of the publishers. Panel (D) Experimental principles of inducible, RNA-guided bottom-up assembly of surface-linked TMV-derived nanotubes spatially directed to surface patches predefined by polymer blend lithography. Subpanel (a) AFM topography image after spin coating a SiO2 with a solution of the two immiscible polymers polystyrene (PS) and polymethyl-methacrylate (PMMA) and demixing of the polymers. Subpanel (b) Same area after selective PS removal by cyclohexane. Uncovered substrate areas are accessible and suitable for bottom-up self-assembly of virus-like nanotubes, as inferred from current models of TMV nucleoprotein assembly. Subpanel (c) Schematic drawing showing uncovered SiO2 patches were functionalized and covalently equipped with DNA linker molecules, the ends of which were then ligated to RNA containing TMV origins of assembly (OAs). Finally TMV CP was added under conditions initiating and sustaining their assembly with RNA. From Mueller et  al. (2011) with permission of the publishers. Panel (E) Diagram for the assembly of nicke- and cobalt-coated TMV1cys templates attached to a gold surface. From Royston et al. (2008) with permission of the publishers. A more detailed version of this figure can be found on http://booksite.elsevier.com/9780123848710.

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with glutamine results in particles exceeding 2  μm in length which contain essentially no RNA and can be metallized in the central channel by Ni (Kadri et al., 2011). Such long “wires” have the potential for electrical connections. Among other uses for modified TMV rods are the formation of field-effect transistors by increasing the number of cycles of ZnO deposition onto TMV nanowires on silicon substrates (Figure 15.14B) (Atanasova et al., 2011) and enhancing the magnetoviscosity of ferrofluids (Wu et al., 2010). Self-assembly of TMV CP in which the N- and C-termini had been reposition to the center of protein assemblies gives disks which are much like those assembled from wild-type protein (Dedeo et al., 2010). The new position of the N-terminus allows functional groups to be installed in the inner pore of the disks giving geometries reminiscent of natural photosynthetic systems. Although TMV particles do not contain any Cys residues that can react with gold, gold nanoparticles bind specifically to the end of the particles most likely by interaction with the RNA (Balci et al., 2007). These metal-virus “nanodumbells” (15.14C) have the potential for establishing nanoscale electrical contacts or, by binding with magnetic metals, could allow manipulation of TMV with external magnetic fields. The ability to self-assemble TMV particles from CP and RNA enables the length of the rods to be controlled by using different sizes of RNA, the sequential and heterologous assembly of CP molecules with different functions (Mueller et  al., 2010) and the formation of various branched structures by using RNA with multiple copies of the OAS (see Chapter 3, Section III, A, 2 for origins of assembly) (Gallie et al., 1987d). There have been several approaches to creating 3-D arrays of modified TMV particles. These include a Cys residue being introduced into the N-terminus of the CP subunits by genetic modification of TMV (Lee et  al., 2005), cDNA which greatly enhances metal deposition on the virus particles and the patterning of TMV particles onto metal surfaces. The particles self-assemble vertically onto gold-patterned substrates due to the surface exposure of the Cys-derived thiol groups at the 3′ end of the TMV rod (Figure 15.14D) (Royston et al., 2008) or possibly by interaction with the viral RNA as suggested above. An anode for a nickel–zinc battery system with enhanced electrode capacity was formed by assembling the modified particles on gold-patterned silica substrates or on a stainless steel surface and then binding nickel onto the bound rods (Royston et  al., 2008; Chen et  al., 2010). Another way of attaching the end of particles to a substrate is by partially disassembling to expose the 5′ end of the RNA which can be bound to virus-specific probe DNA linked to electrodeposited substrate (Figure 15.14E) (Yi et al., 2005; Mueller et al., 2010).

4. Discussion From the above examples it is obvious that there is great potential for the use of plant viruses in nanotechnology, but detailed description is beyond the scope of this book. Current research is focusing on just a few viruses that show different properties. Steinmetz et  al. (2008) compare some of properties of CPMV and TMV for forming layer-by-layer assembly of viral nanoparticles and polyelectrolytes. They conclude that the shape of the viral nanoparticles (VNPs) influences the overall structure of the arrays with CPMV giving alternating arrays of VNPs whereas TMV VNPs are excluded from the arrays and floated on top of the architecture in an ordered structure. However, as is apparent from other chapters in this book, there are many viruses with unique features and it is likely that, at least, some of these will be exploited in future developments of the application of plant viruses to nanotechnology.

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Chapter | 15  Plant Viruses and Technology

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Chapter | 15  Plant Viruses and Technology

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