REVIEW
GM Crops 2:1, 24-33; January/February/March 2011; © 2011 Landes Bioscience
Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts Vincenza Ilardi* and Elisa Di Nicola-Negri CRA-Centro di Ricerca per la Patologia Vegetale; Rome, Italy
Key words: PPV, sharka, Prunus spp., PDR, RNA silencing, ihpRNA, amiRNA, scFv, proteinase inhibitors
Plum pox virus (PPV), a Potyvirus, is the causal agent of sharka, the most detrimental viral disease affecting stone fruit trees. This review focuses on research carried out to obtain PPVresistant transgenic plants and on how biotechnological strategies evolved in light of the scientific advances made during the last several years. Successful RNA silencing strategies that confer a high level of resistance to strains of PPV have been developed and tested under laboratory and greenhouse conditions. Moreover, field tests showed that transgene-mediated RNA silencing was effective in protecting plum plants against aphid-mediated PPV infection. The new emerging biotechnological approaches for conferring PPV resistance are discussed.
efforts are being made to eradicate it or to contain its spread (e.g., EPPO and NAPPO).9,10 The actual strategies to control PPV are based on the use of certified PPV-free plant material, periodic surveys of orchards and eradication of diseased trees. The reduction of aphid vector in orchards by insecticide treatment is not effective against non-persistent viruses such as PPV.11,12 The development of PPV-resistant stone fruit cultivars appears to be the most effective approach to achieve longterm control of PPV and thus to sharka. Unfortunately, few sources of PPV resistance have been identified in stone fruits.13 Despite screening efforts, PPV-resistance was not found in peach, P. persica, germplasm but only in the closely related species P. davidiana clone P1908.14-16 In apricot, P. armeniaca and European plum, P. domestica, some resistance or tolerance traits been identified and breeding programs are carried out.17-22 ©201 1L andeshave Bi os c i enc e. However, the selection process is slow and difficult because of Introduction Donotdi s t r i but e. the undesirable characteristics carried by resistant plants and Stone fruits, Prunus spp., are appreciated worldwide due to their other constraints typical of stone fruit trees such as a long bioorganoleptic characteristics and nutritional value. World produc- logical cycle with an extended juvenile period of seedlings and tion of peaches, nectarines, apricots, plums, sloes and cherries in high degree of heterozygosis.17,23,24 A promising alternative to overcome these constraints is to 2009 was estimated 35 million tons, an increase of 32% since genetically transform the PPV-susceptible elite cultivars and 2000.1 One of the most detrimental diseases affecting stone fruits rootstocks with the opportune resistance gene. This article aims to review the research carried out to obtain is plum pox, also known as sharka. It causes severe fruit symptoms such as malformation, ringspots and in some cases pre- PPV-resistance by genetic engineering and to show how the mature drop resulting in reduced fruit quality and yield.2 The research evolved from the 1990’s up to date in light of the sciendisease was first observed in Bulgaria in the early 1900’s and then tific advances made during the last 20 years. described as a viral disease by Atanassov in 1932.3,4 Thereafter, PPV Genome sharka has spread progressively to a large portion of Europe, around the Mediterranean basin and currently it is widespread in most stone fruit producing countries worldwide except Australia, Organization and expression. PPV genome organization and New Zealand and South Africa (reviewed in ref. 5 and 6). In expression is presented first in order to better illustrate the strategies used by different laboratories to obtain PPV-resistance by 2010, the disease was reported from Japan.7 The causal agent of sharka is Plum pox virus (PPV), which is a genetic engineering. PPV posses a single stranded positive sense RNA genome member of the genus Potyvirus in the family Potyviridae. PPV is transmitted by aphids in a non-persistent manner and by grafting. (Fig. 1) with an average length of 9,770 nucleotides (nt) encapIt is not transmitted vertically through seeds.8 The virus spreads sidated by about 2,000 copies of a single coat protein (CP) in both locally, if infected plants are present in orchards, and over a a flexuous, rod-shaped particle.25 The genomic RNA carries long distance by using infected vegetative propagation material. a covalently linked virus-encoded protein (VPg) at its 5' end In many countries PPV has quarantine status and considerable and poly (A) tail at its 3' end.26,27 The genome contains a long open reading frame (ORF) that is translated as a large polypro*Correspondence to: Vincenza Ilardi; Email:
[email protected] tein precursor from the second AUG codon, leaving 5' and 3' Submitted: 12/30/10; Revised: 02/02/11; Accepted: 02/08/11 untraslated regions (UTRs) of about 150 and 220 nt in length, DOI: 10.4161/gmcr.2.1.15096 respectively.27,28 Cap-independent translation appears mediated
24
GM Crops
Volume 2 Issue 1
REVIEW
REVIEW
©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 1. Genomic map of Plum pox virus (PPV), functions of Potyvirus proteins and schematic representation of PPV sequences utilized for obtaining transgenic PPV-resistant plants. The viral ORF is shown as a box divided into different proteins, named accordingly. PIPO, derived from a frame-shift on the P3 cistron, and P3N-PIPO are indicated as two separated boxes. The gray arrows show cleavage sites recognized by the indicated proteinases. Green T and Rec indicate the recombination sites of PPV-T and PPV-Rec strains respectively. Some known functions of Potyvirus proteins are correspondingly listed above the PPV map. PPV sequences utilized to obtain transgenic PPV-resistant plants are schematized as horizontal lines below the PPV map. The lines orange and light blue portions correspond to the PPV 5' and 3'UTR regions, respectively. White asterisks on lines indicate mutation sites.
by the 5'UTR 29 and VPg.30-32 The PPV polyprotein undergoes proteolytic processing catalyzed by three viral-encoded proteinases,33-35 P1, helper-component proteinase (HC-Pro) and proteinase domain of nuclear inclusion protein a (NIa-Pro). The concerted action of P1, HC-Pro and NIa-Pro proteases give rise to at least ten functional protein products.5,36,37 Moreover, a new ORF (PIPO) resulting from a frameshift in the P3 cistron was reported in the genus Potyvirus (Fig. 1).38 Most, if not all, potyviral proteins are multifunctional and participate in multiple aspects of the viral life cycle (Fig. 1). The following is a brief presentation of protein functions: PPV P1, beside its activity in processing the polyprotein, has an important role in host range specificity.39,40 Moreover, in other Potyvirus, it was shown to possess RNA-binding activity,41-43 and to function in trans as an accessory factor for genome amplification.44 Last, but not least, potyviral P1 was shown to enhance the RNA silencing suppression activity of HC-Pro45,46 (see Box 1 and Fig. 2 for plant RNA silencing description) and to be involved in virus synergism.47
www.landesbioscience.com
The cysteine proteinase HC-Pro has been studied intensively in several Potyvirus. It is involved in aphid transmission,48-50 genome amplification,51 short-52 and long-distance movement,51 symptomathology and pathogenicity,53,54 virus synergism47,55-57 and suppression of RNA silencing-mediated host defense response (Box 1 and Fig. 2).57-62 The N-terminal part of the PPV HC-Pro is indispensable for aphid transmission, the central region is involved in silencing suppression, in maintenance of genome amplification and synergism with other viruses, while the C-terminal part of the protein, known to be necessary mainly for proteolytic activity, also participates in silencing suppression.63 The P3 protein is involved in Potyvirus amplification64,65 and pathogenicity.66,67 Extensive studies on PPV have shown that key host-specific pathogenicity determinants lie in a region that spans P1, HC-Pro, P3 and 6K1 peptides.40,66-68 The protein product of the newly identified potyviral ORF called P3N-PIPO was recently shown to participate in movement of Potyviruses.69,70 CP, other than the cylindrical inclusion (CI) and P3N-PIPO proteins, is required for viral cell-to-cell
GM Crops
25
movement.52,71-73 CI protein, which posses NTPase and helicase PPV resistance in herbaceous model plants. Most biotechactivities, is also required for PPV genome replication74-77 together nological strategies developed to control PPV infection are based with the nuclear inclusion protein b (NIb) that is the potyviral on expression of viral-derived sequences in plants. In particular, RNA-dependent RNA polymerase (RdRp).78,79 The replication from the early 1990’s up to the discovery of RNA silencing pheof Potyviruses in infected cells is associated with intracellular nomenon (Box 1 and Fig. 2), and its potential use as a tool to membranous vesicles. In particular, 6K2 protein induces the for- obtain virus-resistant plants, the leading idea to induce PPV resismation of endoplasmic reticulum (ER)-derived vesicles.80-83 tance was to express PPV-derived proteins in plants. This general Finally, the C-terminal product of the precursor polyprotein is idea was based on the assumption that constitutive expression of the capside protein (CP). The potyviral CP posses a central con- selected wild type or opportunely mutagenized viral proteins in served core domain and variable N- and C-terminal domains. The plant could interfere with virus replication. conserved core domain is necessary for virion assembly, cell-toDue to the proteolytic origin of PPV proteins, their ectopic cell and long-distance movement.71,72,84 The terminal domains are expression in plants require the introduction of extra start and exposed on the surface of the virion and in particular the N-terminal stop codons as well as UTR regions at the 5' and 3' ends of the domain, beyond its role in viral long distance movement, contains selected ORF, respectively.84,96-104 the conserved DAG aminoacid triplet required for the interaction The PPV-derived sequences expressed in plants (Fig. 1) are: with HC-Pro-bridge protein for aphid transmission.50 (a) the CP gene; 84,96-98,102,103,105-107 (b) the NIb gene, that was introStrains. PPV isolates with specific pathogenicity, symptom- duced into N. benthamiana genome either alone99 or in associaatology, host range, epidemiology and aphid transmissibility tion with the NIa and the 3'-terminus sequence of the CP;100 (c) are continuously collected worldwide. On the basis of molecu- the proteinase P1;108 (d) the CI;101 (e) the HC-Pro.104 Moreover, lar and serological properties PPV isolates have been grouped based on the assumption that CP resistance was protein-mediinto seven strains: D, M, Rec, EA, T, W and C.85-90 D and M ated some groups expressed in plants opportunely modified PPV are the most widespread and economically important strains. CP versions to control the biological risk of hetero encapsidation PPV-Rec is reported mainly from Europe, PPV-EA is confined and aphid transmission of the hetero encapsidate virions.84,102,109 to Egypt, PPV-T to Turkey and PPV-W to Canada (PPV-W has Although all these plants were engineered to express different been declared eradicated10). Finally, PPV-C is a sour and sweet PPV protein products, from a mechanistic resistance point of cherry-adapted strain reported mainly from Europe. PPV-Rec view, they can be substantially treated all together. In fact, a first and PPV-T are characterized by recombination events aspect ©20between 1 1L andescommon Bi os c i en c e. was that only a minor fraction of the transgenic PPV-M and PPV-D genomes (Fig. 1), while PPV-EA, PPV-C plants showed PPV resistance and most importantly that resisDonotdi s t r i but e. and PPV-W are very highly divergent strains suggesting that they tance was not positively correlated with the amount of transgenic constitute distinct independent evolutionary lineages.91 Although protein.96,98-101,103,104,108-110 This situation is unexpected if the resisPPV isolates can be grouped based on their molecular and sero- tance mechanism is protein-mediated. In several PPV-resistant logical features the biological properties of PPV isolates seem plants transgenic proteins were undetectable, while in other cases, to depend more on isolate-specific characteristics than on their an inverse correlation between the amount of transgenic mRNA taxonomic status. However, some strain’s specific biological char- and/or protein and the resistance was observed.98-101,104,108,109 acteristics are: PPV-C isolates appear to be the only ones that Furthermore, several transgenic lines showed recovery from viral infect cherry plants while PPV-M isolates usually spread more infection. In those transgenic plants, PPV symptoms were inireadily by aphids and cause more severe symptoms on peach than tially apparent but as plants grew they progressively disappeared PPV-D isolates.91 and the virus became undetectable on the newly emerging leaves, moreover, the accumulation of transgene transcripts showed a Transgenic Strategies to Control PPV Infection drastic reduction when analyzed.84,96,99,108,109 All these findings indicate that in those PPV-resistant plants, the resistant phenoThe biotechnological strategies developed to control plant viruses type was not protein-mediated but RNA-mediated.98-100,104,111,112 and spread of viral diseases may be classified into three major Retrospectively, it is now possible to explain most, if not all, precategories based on the type of nucleic acid sequences used. The vious data on PPV resistance as a result of the unwanted and first comprises those strategies that make use of sequences derived unpredictable activation of the degradative RNA silencing pathfrom the viral genomes; a concept initially proposed by Sanford way (Fig. 2 and Box 1). It is now accepted that one reason for & Johnston and known as pathogen-derived resistance (PDR).92 the unpredictable activation of the RNA silencing pathway in The second involves plant derived genes (e.g. see ref. 93) and the plants expressing transgenic RNA sequences is the integration, in third is non-viral, non-plant derived sequences (e.g. see ref. 94). the host genome, of multiple and/or rearranged T-DNA copies Before describing what was done to obtain resistance to PPV, which in turn lead to transcription of aberrant or double stranded it is important to note that because transformation and regenera- RNAs (dsRNA), the inducers of RNA silencing mechanism.113-116 tion of fruit trees are demanding and time consuming,95 most, Taking into account that dsRNA is the inducer of RNA silencif not all, PPV resistance strategies have been previously tested ing, then it was possible to opportunely construct a transgene to on herbaceous model plants. Hence, most available data on PPV express virus-derived dsRNAs in plants leading to a pre-activated resistance come from studies conducted with Nicotiana spp. host resistance mechanism against any incoming viruses pos(mainly N. benthamiana) that are model plants in virus research. sessing genome sequence homologues to the transgene. Smith et
26
GM Crops
Volume 2 Issue 1
Figure 2. Simple and schematic illustration of post-transcriptional gene silencing (PTGS) in plants (for detail see Box 1). (A) PTGS by small interfering RNA (siR©201 1L and espolymerase; Bi os c i enc e.ovals represent RNA polymerase II; orange ovals represent NA). (B) PTGS by microRNA (miRNA). Ochre oval represents RNA-dependent RNA gray Dicer-like dsRNA-specific RNase III ribonucleases; pink ovals represent RNA methyltransferase Don otd i s t r i but e. HEN1; light blue ovals represent Argonaute proteins present in RNA-Induced Silencing Complexes (RISC); green ovals represent ribosomal subunits; yellow circle represent Potyvirus HC-Pro RNA silencing suppressor. Box 1. RNA silencing refers to a family of gene silencing phenomena triggered by double-stranded RNA (dsRNA) molecules by which the expression of nucleic acid sequences is downregulated or entirely suppressed.113-116 The different plant RNA-silencing pathways show four conserved steps: (A) induction by dsRNAs or self-complementary foldback RNAs, (B) dsRNA processing into small RNAs (sRNAs) of 18–25 nucleotides in length by Dicer-like proteins, (C) methylation of sRNA by HEN1 methyltransferase and (D) sRNA incorporation into RNA-induced silencing complexes (RISC) that associate with partially or fully complementary target RNA or DNA (Fig. 2). The two major classes of sRNAs are the small interfering RNAs (siRNAs) and the microRNAs (miRNAs). dsRNAs may derive directly from highly structured single-stranded viral RNAs, virus replication, transcription of endogenous loci producing transcripts with internal stem-loop structures or transgenic inverted-repeat sequences. Alternatively, dsRNA may be synthesized by one of six RNA-dependent RNA polymerases (RdRp) that copy single-stranded RNA (ssRNA) possessing “particular” features. Upon processing by one Dicer-like protein, that is a dsRNA-specific RNase III ribonuclease, sRNA duplexes could be either retained in the nucleus for transcriptional gene silencing (TGS) or exported to the cytoplasm for post-transcriptional gene silencing (PTGS). A selected sRNA strand incorporates into one or more RISCs, which is the generic name for an Argonaute-sRNA complex (it may also contain auxiliary proteins), that scan the cell for complementary nucleic acids to perform its functions. In plants sRNA-directed RISC functions include: (A) RNA endonucleolytic cleavage in the middle of sRNAtarget hybrids (in siRNA and miRNA pathways for PTGS, Fig. 2A and B), (B) repression of translation (in miRNA pathway for PTGS, Fig. 2B), (C) DNA cytosine and/or histone methylation (in siRNA pathway for TGS, not shown). It is difficult to distinguish between miRNA and siRNA in plants, the only reliable criterion is that miRNAs are excised as discrete species from the stem of an imperfect stem-loop precursor (pre-miRNA), whereas siRNAs occur as populations produced from perfect or near-perfect RNA duplexes.147,148 In plants RNA silencing, among other functions, is involved in the defense against viruses. Viruses, as counter-defense have evolved proteins that interfere/suppress one or more steps of the RNA silencing pathway.149-151 In the genus Potyvirus the protein HC-Pro acts as an RNA silencing suppressor that binds siRNAs preventing their loading into RISC (Fig. 2A). 36,47,60,62
al. showed that gene constructs encoding intron-spliced hairpin RNAs (ihpRNAs) can induce RNA silencing with high efficiency (>90%) opening the way on how to effectively apply RNA silencing technology to obtain virus-resistant plants.117 In order to develop robust tools for inducing PPV resistance, that are not highly dependent on a particular and uncontrolled event of transformation, PPV-derived constructs based on the ihpRNA technology117 were developed using isolates of PPV-D118-121 or -M strain.122 Pandolfini et al. utilized a short
www.landesbioscience.com
DNA sequence covering the polyprotein AUG translation initiation codon (Fig. 1) placed as two inverted repeats separated by rolA intron, under the transcriptional control of the rolC promoter. The rolC promoter drives transgene expression in phloem but not in epidermal and mesophyll cells. Resistance to systemic PPV-D infection was obtained in N. benthamiana plants while local infection was unaffected as expected by the phloem specificity of the promoter. Due to PPV-D accumulation in inoculated leaves a complex trait of PPV-D
GM Crops
27
resistance/susceptibility was observed. However, PPV-D sys- underline the importance to select the appropriate promoter, tertemic resistance was observed in a consistent fraction of the minator and intron as well as the length and the type of viral progeny of all independent transgenic lines analyzed. sequences to build up ihpRNA constructs that confer high and In the work of Di Nicola-Negri et al.122 four ihpRNA con- broad-spectrum PPV resistance. structs covering the 5'UTR, P1, HC-Pro and P3 sequences Beside PDRs, the other biotechnological strategy developed (Fig. 1) of PPV-M ISPaVe44 were introduced in N. benthami- and tested in N. benthamiana belongs to the non-plant, nonana plants. In this case the transcription was driven by the 35S pathogen derived class. In particular, this strategy was conceived Cauliflower mosaic virus (CaMV) promoter and the intron was to interfere with PPV NIb or CP functions by expressing in from Arabidopsis thaliana Dof Affecting Germination (DAG1) plants single-chain variable fragment (scFv) antibodies specific to gene. Thirty-eight out of 40 T0 plants (95%) did not show any these proteins.124 At 30 dpi in the best performing scFv/NIb and detectable accumulation of PPV-M ISPaVe44 when analyzed in a scFv/CP lines plants positive for PPV-D/NAT were only 17% leaf disk assay. Two lines for each construct were further analyzed. and 21%, respectively. Although this line of research was not Two hundred forty eight out of 253 T1 plants (98%) showed further developed, this methodological approach was shown feacomplete resistance to local and systemic PPV-M ISPaVe44 infec- sible to confer resistance to multiple viruses;125 hence, scFvs direct tion. Importantly, the h-UTR/P1 construct, covering the PPV against conserved domain of non-structural viral proteins could 5'UTR and a part of the P1 gene, was shown to confer long last- be another tool for efficiently interfering with viral infection. ing resistance to nine PPV isolates of the D, M, Rec, EA and C From herbaceous model plants to stone fruit GM crops. strains originally collected from peach, plum, apricot, almond The first PPV constructs introduced in stone fruit plants were and sweet cherry in different geographical areas.123 The above those expressing the sense CP gene; in particular transgenic results have made the h-UTR/P1 construct of particular prac- apricot, P. armeniaca cv. Kecskemeter,105 and European plum, tical interest to obtain transgenic stone fruit plants resistant to P. domestica cv. Stanley,107 were produced. Among transgenic PPV. stone fruit clones obtained, only plum C5 was resistant to PPV Hily et al.120 reported that four ihpRNA constructs, harboring under greenhouse conditions.110 C5 is characterized by possessthe full length or the second half of the CP gene (Fig. 1), under ing multiple and rearranged CP gene copies. It produces a low the transcriptional control of either CaMV 35S or peach Cab pro- level of CP mRNA and does not accumulate a detectable amount moters, were introduced in N. benthamiana plants. Among the of CP.107,112 Moreover, methylation of the PPV CP insert as well 126 48 transgenic T0 lines obtained, 37 (77%) accumulated detectCP specific ©201 1 L andesas Bi os c i enc e.siRNAs were observed. As discussed above, all able CP-specific siRNA. At 60 days post inoculation (dpi) 54% these molecular features are characteristics of the unpredictable Donotdi s t r i but e. of the T1 progeny derived from 13 siRNA producing T0 lines activation of post-transcriptional transgene silencing. C5 clone were resistant to PPV-D PENN3 infection whereas T1 progeny was extensively tested in field to validate its ability to resist PPV of siRNA not expressing lines were all infected. No difference infection under natural environments. Plantings were made in in the percentage of PPV-resistant plants was observed between Poland, Spain, Romania and the Czech Republic at sites charlow or high siRNA producing lines. However, the full-length acterized by sharka presence.127,128 Field tests showed that the C5 ihpRNA CP construct was shown to outperform the shorter one plants were resistant to PPV when exposed to natural viruliferous in conferring PPV resistance. aphids while those PPV-inoculated by either chip bud-, bark- or Liu et al.119 reported the production of an ihpRNA construct rootstock-infected plant material accumulated a low level of PPV, created by fusion of gene fragments from six stone fruit infect- mostly near the graft junction. In Poland PPV was detected on ing viruses including a portion of PPV-D CP gene (Fig. 1). The both C5 grafted plants one year later than the control; in the idea behind this approach was to build a single construct that Czech Republic the PPV titer and symptoms in C5 plants were interferes with most important stone fruit infecting viruses. Of reduced over a three-year period.127,129 In Poland and Spain, the twenty-eight transgenic N. benthamiana lines two T3 homo- the C5 trees were challenged with PPV-D while in the Czech zygous ones were further selected for multi-virus resistance tests. Republic with PPV-Rec. Moreover, to test the effect of heteroloT3 plants challenged with PPV-D isolate did not show any visible gous viruses infection on the efficacy and stability of C5 PPV resistance, C5 plants were graft-inoculated with different combisymptoms. Finally, Wang et al.121 developed two ihpRNA constructs from nations of Prunus necrotic ringspot virus (PNRSV), Apple chlorotic a Canadian PPV-D isolate using the 5' portion of P1 and the 3' leaf spot virus (ACLSV), Prunus dwarf virus (PDV) and PPV-D129 portion of CP genes, respectively. Differently from the strategy or PPV-Rec.128 Collectively, these analyses confirmed that a low used by Di Nicola-Negri et al.122 in this case the UTR sequences level of PPV could only accumulate in C5 grafted plants and that were not included in the ihpRNA constructs (Fig. 1). The tran- heterologous virus infection sustained by PNRSV, ACLSV or scription was driven by the CaMV 35S promoter and the intron PDV did not suppress PPV resistance. The lack of complete PPV was from peach endo-polygalacturonase (endo-PG) gene. PPV-D resistance in graft-inoculated C5 plants may be due to the high resistance was observed in 49% of P1- and 41% of CP-derived and continuous viral inoculum source. Conversely, aphids, the transgenic N. benthamiana lines. natural vectors of PPV, are mostly present only at a defined and Collectively, PPV resistance data of ihpRNA transgenic restricted period of time during the year. N. bethamiana plants clearly show the feasibility of this technolBeside the C5 clone, plum plants resistant to PPV were ogy to efficiently control PPV infection. Moreover, they clearly recently obtained using the ihpRNA technology.120,121 Hily et
28
GM Crops
Volume 2 Issue 1
al.120 transformed P. domestica cv. Stanley with the CaMV 35S full-length ihpRNA CP construct that was shown to outperform the shorter ihpRNA CP in conferring PPV resistance in N. benthamiana plants. One plum clone, ihpRNA-B14, accumulating CP-specific siRNAs was micropropagated and analyzed for PPV-D resistance. Sixteen months after aphid inoculation, the seven tested plants did not accumulate viral CP as evaluated by DAS-ELISA and RT-PCR. Similarly, Wang et al.121 transformed Stanley plums with ihpRNA P1 construct that gave the best resistance results in the model plant. One to six plants for each transgenic line were challenged by PPV-D infected chip-buds. Importantly, PPV was undetectable in five out of the ten T0 transgenic lines analyzed. Finally, PPV-M derived h-UTR/P1 construct, capable to confer resistance to PPV isolates of D, M, Rec, EA and C strains in N. benthamiana plants,123 was recently introduced in Stanley and Tardicotes plum varieties. Two transgenic clones were challenged with a PPV-D isolate by in vitro grafting. More than 95% of tested plants of clone ST24 were resistant (Damiano and Ilardi, unpublished results). The above results, although still not conclusive, underline a good correlation between PPV ihpRNA resistance data obtained on N. benthamiana and those gathered on stone fruit crops, bearing out the importance of conducting initial studies on herbaceous model plants.
viruses.131 Moreover, the very low RNA steady-state level of transgenic transcripts as well as their molecular structure (dsRNAs) may reduce the likelihood, if any, of a hypothetical unwanted recombination between the ihpRNA and the RNA genome of an incoming virus. A point that remains to be further investigated is that at what extent biotic and abiotic factors known to have some impact on RNA silencing132,133 could interfere with ihpRNA-mediated PPV resistance. However, studies conducted on the plum C5 clone suggested that, at least in that case, natural biotic and abiotic conditions do not suppress transgenic PPV resistance.127-129 Beside the ihpRNA-mediated RNA silencing other technologies are fast emerging. It is expected that in the near future they will be applied with success for conferring PPV resistance. In this context it has been recently shown that PPV chimeras harboring wild type but not mutated miRNA target sequences have an impaired infectivity when inoculated on wild type N. benthamiana and A. thaliana plants134 and that plant miRNA precursors can be engineered to express, in transgenic plants, artificial miRNAs (amiRNAs) (Fig. 2) complementary to viral genomes.135 A. thaliana plants engineered to express amiRNAs complementary to P69 of Turnip yellow mosaic virus (TYMV) and HC-Pro of Turnip mosaic virus (TuMV) gene sequences were specifically resistant to TYMV and TuMV, respectively.135 Similarly, amiRNAs targeting the 2b gene or the 3'UTR of Cucumber mosaic virus (CMV) conferred resistance to CMV infection.136,137 As design Concluding Remarks and Future Perspectives ©201 1L andesamiRNA Bi os c i en c e. requires only a stretch of 20–24 nt complementary to the target, it should be possible, in principle, to select Donotdi s t r i but e. Many efforts have been made worldwide to obtain stone fruit sequences that efficiently target viral RNAs without having offtrees resistant to PPV. As pointed out above, transgenic technol- target effects.138 Off-target effects refer to unintended host gene ogy has been shown to be effective in producing stone fruit plants regulation effects that may occur as a result of sequence homolresistant to PPV.112,120,121 The evidence that dsRNA, the inducer ogy between the ectopically expressed small RNAs (siRNA, of RNA silencing, can efficiently confer virus resistance in her- amiRNA) and mRNAs of the recipient organism. It is important baceous model plants118,120-122 together with the demonstration to underline that biosafety concerns about potential recombithat RNA silencing operates in transgenic plum to restrict PPV nation events between ectopically expressed pathogen-derived infection112 have opened the way to build new refined ihpRNA sequences and unrelated viruses do not apply to the amiRNA constructs to confer predictable PPV resistance in stone fruit technology. Moreover, a not fully explored advantage of using plants120,121 (Damiano and Ilardi, unpublished results). Although amiRNAs respect of ihpRNAs is that amiRNA activity, at least RNA silencing operates in a sequence-specific fashion, the data in the model plant A. thaliana, is not affected135 by temperatures obtained on transgenic plants transformed with PPV-derived (i.e., 15°C) known to have detrimental effect on hpRNA-mediihpRNA constructs clearly show that it is possible to identify ated silencing.133 Nevertheless, it should be noted that PPV resisviral genomic regions that confer a broad spectrum of resistance tance was not broken in the 5'UTR/P1 ihpRNA transgenic N. to most, if not all, known PPV strains.123 Moreover, two aspects benthamiana plants grown at 15°C (Di Nicola-Negri and Ilardi of ihpRNA technology should be pointed out. First, the ihpR- unpublished results) whereas the level of the artificial miR2b NAs induce consistent PPV resistance in a high number of the in agro-infiltrated N. benthamiana leaves was reduced at 15°C initially transformed herbaceous and stone fruit plants.121,122 This compared to that at 25°C.136 Thus additional work is required to aspect is of particular interest considering the low efficiency in better clarify the impact of low temperatures on amiRNA- and obtaining transgenic stone fruits. In fact, although methods are ihpRNA-derived virus resistance. Conversely, whereas molecucontinuously improved,95,130 in most stone fruit species transfor- lar evolution of viral genomes under selective pressure of amiRmation and regeneration of commercial varieties are limited to NAs could be envisaged as a potential Achille’s heel, this aspect few genotypes and the transformation/regeneration efficiency is clearly assumes much less importance with respect to standard far from that obtained in herbaceous plants. The second aspect ihpRNA technology (dsRNA longer than 400 nt). In this context with regard to biosafety, RNA silencing technology does not a recent work has shown that if viral replication is permitted, require the expression of transgenic proteins, thus circumvent- RNA viruses quickly evolve breaking down amiRNA-mediated ing possible risk of allergy and/or toxicity for human and animal resistance.139 Hence, ihpRNAs appear, in principle, more suitable and of complementation and/or synergy on behalf of incoming than amiRNAs for conferring long lasting broad-spectrum virus
www.landesbioscience.com
GM Crops
29
resistance. However, we believe that in the near future a refined expression of a rice cysteine proteinase inhibitor orycystatin in amiRNA-mediated approach together with the ihpRNA technol- N. tabacum interferes with TEV and Potato virus Y (PVY) infecogy, may contribute in obtaining PPV-resistant stone fruit trees. tion; a direct correlation between oryzacystatin message levels, The comprehension of the molecular basis of how viruses inhibition of papain and resistance to the viruses was observed. not only exploit but can also suppress plant endogenous path- In vitro assays have been shown that cystatins, of plant or human ways is opening the way for the development of novel strate- origin, inhibit, with low efficiency, the activity of PPV proteingies to control viral infection. Among host-encoded factors ases.33,144 Although an efficient inhibitor of PPV proteinases has required for PPV infection the eukaryotic translation initiation not been isolated other approaches like scFv145 or the expression factor eIF(iso)4E has been shown to play an important role. of dominant interfering peptides146 could be profitably used to Interestingly, disruption of the eIF(iso)4E gene by transposon interfere with the proteolytic processing. tagging does not lead to any developmental modifications of Beside the efforts to identify and develop the best molecular A. thaliana plants while it confers resistance to five PPV iso- strategies to effectively control PPV, consequently, sharka dislates of the D, M, Rec, EA and C strains.140,141 Recently, Huang ease, other transversal research activities should be conducted on: et al.142 have isolated two DEAD-box RNA helicase-like pro- (a) improvement of stone fruit transformation and regeneration teins, PpDDXL and AtRH8 from peach and Arabidopsis, process; (b) development of methods to avoid use of antibioticrespectively, both interacting with PPV VPg. They showed dependent selection of transformants or to allow elimination of that AtRH8 is dispensable for plant growth and development marker genes from transformed plants; (c) identification and isobut necessary for PPV infection. Transient overexpression of lation of stone fruit species specific regulative sequences, such as the VPg-binding region from either AtRH8 or PpDDXL sup- promoters, terminators and introns, to regulate the expression of presses TuMV and Tobacco etch virus (TEV) accumulation in transgenes avoiding where not strictly necessary the use of noninfected N. benthamiana leaf tissues. Taken together, these plant derived sequences. data suggest that AtRH8, PpDDXL as well as other host facAcknowledgements tors necessary for PPV pathogenicity could be manipulated for the development of genetic resistance to PPV. Finally, the We are grateful to Mario Tavazza for critical reading of the manbiochemical interference with the proteolytic maturation of the uscript and his suggestions, anonymous reviewers for improving viral polyprotein, could be another approach to obtain PPV the article and Agricultural Research Council (CRA) for financonstitutive support through the SHARE project. resistance. Gutierrez-Campos et al.163 showed that©2 01 1L andescial Bi o s c i enc e . References 1.
FAOSTAT. Food and Agriculture Organization of the United Nations 2011. Availalbe from: http://faostat. fao.org. 2. Llacer G, Cambra M. Hosts and symptoms of Plum pox virus: fruiting Prunus species. EPPO Bull 2006; 36:219-21. 3. Atanasoff D. Plum pox. A new virus disease. Annals of the University of Sofia, Faculty of Agriculture and Silviculture 1932; 11:49-69. 4. Németh M. Virus, mycoplasma and rickettsia diseases of fruit trees. Martinus-Nijhoff Publishers, Dordrrecht, Holland 1986; 841. 5. García JA, Cambra M. Plum pox virus and sharka disease. Plant Viruses 2007; 1:69-79. 6. Barba M, Hadidi A, Candresse T, Cambra M. Plum pox virus. In: Hadidi A, Barba M, Candresse T, Jelkmann W, ed. Virus and Virus-Like Diseases of Pome and Stone Fruits. St. Paul MN: The American Phytopathological Society Press, 2011:In press. 7. Maejima K, Hoshi H, Hashimoto M, Himeno M, Kawanishi T, Komatsu K, et al. First report of Plum pox virus infecting Japanese apricot (Prunus mume Sieb. et Zucc.) in Japan. J Gen Plant Path 2010; 76:229-31; DOI: 10.1007/s10327-010-0233-6. 8. Pasquini G, Barba M. The question of seed transmissibility of Plum pox virus. EPPO Bull 2006; 36:287-92. 9. EPPO A2 List of pests recommended for regulation as quarantine pests. EPPO (version 2010-09) Available from: http://www.eppo.org/QUARANTINE/listA2. htm 10. North American Plant Protection Organization's Phytosanitary Alert System. NAPPO 2010; Available from: http://www.pestalert.org 11. Perring TM, Gruenhagen TM, Farrar CA. Management of plant viral diseases through chemical control of insect. Annual Rev Entomol 1999; 44:457-81.
30
12. Carraro L,D Labonne G,d Dallot S.u Vecor control of fruit onot i s t r i b t e. tree viruses and phytoplamasmas. In: Hadidi A, Barba M, Candresse T, Jelkmann W, ed. Virus and Virus-Like Diseases of Pome and Stone Fruits. St. Paul, MN: The American Phytopathological Society Press, 2011:In press. 13. Decroocq M, Badenes L, Neumüller M. Breeding for resistance to Plum pox virus. In: Hadidi A, Barba M, Candresse T, Jelkmann W, ed. Virus and Virus-Like Diseases of Pome and Stone Fruits. St. Paul MN: The American Phytopathological Society Press, 2011:In press. 14. Kervella J, Pascal T, Pfeiffer F, Dirlewanger E. Breeding for multiresistance in peach tree. Acta Hort 1998; 465:177-84. 15. Marandel G, Pascal T, Candresse T, Decroocq V. Quantitative resistance to Plum pox virus in Prunus davidiana P1908 linked to components of the eukaryotic translation initiation complex. Plant Pathol 2009; 58:425-35. 16. Rubio M, Pascal T, Bachellez A, Lambert P. Quantitative trait loci analysis of Plum pox virus resistance in Prunus davidiana P1908: new insights on the organization of genomic resistance regions. Tree Genet Gen 2010; 6:291-304. 17. Kegler H, Hartman W. Present status of controlling conventional strains of Plum pox virus. In: Hadidi A, Khetarpal RK, Koganezawa H, ed. Plant Virus Disease Control. St. Paul, MN: APS Press, 1998:616-28. 18. Marandel G, Salava J, Abbott A, Candresse T, Decroocq V. Quantitative trait loci meta-analysis of Plum pox virus resistance in apricot (Prunus armeniaca L.): new insights on the organization and the identification of genomic resistance factors. Mol Plant Pathol 2009; 10:347-60. 19. Karayiannis I, Thomidis T, Tsaftaris A. Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L). Tree Genet Genome 2008; 4:143-8.
GM Crops
20. Hartmann W, Neumüller M. Plum breeding. In: Breeding Plantation Tree Crops. Temperate Species. Jain SM, Priyadarshan PM, ed. Berlin, Germany: Springer, 2009:161-232. 21. Soriano JM, Vera-Ruiz EM, Vilanova S, MartinezCalvo J, Llacer G, Badenes ML, et al. Identification and mapping of a locus conferring Plum pox virus resistance in two apricot-improved linkage maps. Tree Genet Genomes 2008; 4:391-402. 22. Dondini L, Lain O, Vendramin V, Rizzo M, Vivoli D, Adami M, et al. Identification of QTL for resistance to Plum pox virus strains M and D in Lito and Harcot apricot cultivars. Mol Breed 2010; 27:289-99; DOI: 10.1007/s11032-010-9431-3. 23. Decroocq V, Foulongne M, Lambert P, Le Gall O, Mantin C, Pascal T, et al. Analogues of virus resistance genes map to QTLs for resistance to sharka disease in Prunus davidiana. Mol Gen Genom 2005; 272:680-9. 24. Ion-Nagy L, Lansac M, Eyquard JP, Salvador B, Garcia JA, Le Gall O, et al. PPV long-distance movement is occasionally permitted in resistant apricot hosts. Virus Res 2006; 120:70-8. 25. ICTVdB Descriptions. International Committee on Taxonomy of Viruses 2002; Available from: http://www. ictvdb.rothamsted.ac.uk/ICTVdB/00.057.0.01.054.htm 26. Riechmann JL, Laín S, García JA. The genome-linked protein and 5' end RNA sequence of plum pox Potyvirus. J Gen Virology 1989; 70:2785-9. 27. Laín S, Riechmann JL, Méndez E, García JA. Nucleotide sequence of the 3' terminal region of plum pox Potyvirus RNA. Virus Res 1988; 10:325-42. 28. Riechmann JL, Laín S, García JA. Identification of the initiation codon of plum pox Potyvirus genomic RNA. Virology 1991; 185:544-52. 29. Kneller EL, Rakotondrafara AM, Miller WA. Capindependent translation of plant viral RNAs. Virus Res 2006; 119:63-75.
Volume 2 Issue 1
30. Michon T, Estevez Y, Walter J, German-Retana S, Le Gall O. The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue. FEBS J 2006; 273:1312-22. 31. Beauchemin C, Boutet N, Laliberte JF. Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of Turnip mosaic virus, and the translation eukaryotic initiation factor (iso) 4E in planta. J Virol 2007; 81:775-82. 32. Khan MA, Miyoshi H, Gallie DR, Goss DJ. Potyvirus genomelinked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F. J Biol Chem 2008; 283:1340-9. 33. García JA, Cervera MT, Riechmann JL, López-Otín C. Inhibitory effects of human cystatin C on plum pox Potyvirus proteases. Plant Mol Biol 1993; 22:697-701. 34. Ravelonandro M, Peyruchaud O, Garrigue L, de Marcillac G, Dunez J. Immunodetection of the Plum pox virus helper component in infected plants and expression of its gene in transgenic plants. Arch Virol 1993; 130:251-68. 35. García JA, Riechmann JL, Laín S. Proteolytic activity of the plum pox Potyvirus NIa-like protein in Escherichia coli. Virology 1989; 170:362-9. 36. Urcuqui-Inchima S, Haenni AL, Bernardi F. Potyvirus proteins: a wealth of functions. Virus Res 2001; 74:157-75. 37. Salvador B, García JA, Simón-Mateo C. Causal agent of sharka disease: Plum pox virus genome and function of gene products. EPPO Bulletin 2006; 36:229-38. 38. Chung BY, Miller WA, Atkins JF, Firth AE. An overlapping essential gene in the Potyviridae. Proc Natl Acad Sci USA 2008; 105:5897-902. 39. Salvador B, Saenz P, Yanguez E, Quiot J, Quiot L, Delgadillo MO, et al. Host specific effect of P1 exchange between two Potyviruses. Mol Plant Pathol 2008; 9:147-55. 40. Salvador B, Delgadillo MO, Sáenz P, Garcia JA, SimónMateo C. Identification of Plum pox virus pathogenicity determinants in herbaceous and woody hosts. Mol Plant-Microbe Interact 2008; 21:20-9. 41. Brantley JD, Hunt AG. The N-terminal protein of the polyprotein encoded by the Potyvirus tobacco vein mottling virus is an RNA-binding protein. J Gen Virol 1993; 74:1157-62. 42. Soumounou Y, Laliberté JF. Nucleic acid-binding properties of the P1 protein of turnip mosaic Potyvirus produced in Escherichia coli. J Gen Virol 1994; 75:256773. 43. Merits A, Guo DY, Saarma M. VPg, coat protein and five non-structural proteins of potato A Potyvirus bind RNA in a sequence-unspecific manner. J Gen Virol 1998; 79:3123-7. 44. Verchot J, Carrington JC. Evidence that the Potyvirus P1 proteinase functions in trans as an accesory factor for genome amplification. J Virol 1995; 69:3668-74. 45. Rajamaki ML, Kelloniemi J, Alminaite A, Kekarainen T, Rabenstein F, Valkonen JP. A novel insertion site inside the Potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions. Virology 2005; 342:88-101. 46. Valli A, Martín-Hernández AM, López-Moya JJ, García JC. RNA silencing suppression by a second copy of the P1 serine protease of Cucumber vein yellowing ipomovirus (CVYV), a member of the family Potyviridae that lacks the cysteine protease HCPro. J Virol 2006; 80:10055-63. 47. Pruss G, Ge X, Shi XM, Carrington JC, Vance BV. Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 1997; 9:859-68. 48. Govier DA, Kassanis B. A virus-induced component of plant sap needed when aphids acquire Potato virus Y from purified preparations. Virology 1974; 61:420-6.
www.landesbioscience.com
49. Govier DA, Kassanis B, Pirone TP. Partial purification and characterization of the Potato virus Y helper component. Virology 1977; 78:306-14. 50. Blanc S, López-Moya JJ, Wang RY, García-Lampasona S, Thornbury DW, Pirone TP. A specific interaction between coat protein and helper component correlates with aphid transmission of a Potyvirus. Virology 1997; 231:141-7. 51. Kasschau KD, Cronin S, Carrington JC. Genome amplification and long-distance movement functions associated with the central domain of tobacco etch Potyvirus helper component-proteinase. Virology 1997; 228:251-62. 52. Rojas MR, Zerbini FM, Allison RF, Gilbertson RL, Lucas WJ. Capsid protein and helper componentproteinase function as Potyvirus cell-to-cell movement proteins. Virology 1997; 237:283-95. 53. Sáenz P, Quiot L, Quiot JB, Candresse T, García JA. Pathogenicity determinants in the complex virus population of a Plum pox virus isolate. Mol Plant Microbe Intereract 2001; 14:278-87. 54. Kasschau KD, Xie ZX, Allen E, Llave C, Chapman EJ, Krizan KA, et al. P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 2003; 4:205-17. 55. Vance VB. Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 1991; 182:486-94. 56. Yang S, Ravelonandro M. Molecular studies of the synergistic interactions between Plum pox virus HC-Pro protein and potato virus X. Arch Virol 2002; 147:2301-12. 57. Gonzalez-Jara P, Atencio FA, Martinez-Garcia B, Barajas D, Tenllado F, Diaz-Ruiz JR. A single amino acid mutation in the Plum pox virus helper componentproteinase gene abolishes both synergistic and RNA silencing suppression activities. Phytopathol 2005; 95:894-901. ©201 1L andesBi os c i enc e. 58. Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau D o n o t d i s t r i b u t e . C, Mallory A, et al. A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 2000; 290:142-4. 59. Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 1998; 17:6739-46. 60. Kasschau KD, Carrington JC. A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 1998; 95:461-70. 61. Tenllado F, Barajas D, Vargas M, Atencio FA, GonzálezJara P, Díaz-Ruíz JR. Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing. Mol Plant-Micr Inter 2003; 16:149-58. 62. Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu YP, et al. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 2006; 25:2768-80. 63. Varrelmann M, Maiss E, Pilot R, Palkovics L. Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the Plum pox virus helper component proteinase suppressor of gene silencing. J Gen Virol 2007; 88:1005-15. 64. Klein PG, Klein RR, Rodríguez-Cerezo E, Hunt A, Shaw JG. Mutational analysis of the tobacco vein mottling virus genome. Virology 1994; 204:759-69. 65. Langenberg WG, Zhang L. Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions. J Struct Biol 1997; 118:243-7. 66. Sáenz P, Cervera MT, Dallot S, Quiot L, Quiot JB, Riechmann JL, et al. Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3 + 6K1. J Gen Virol 2000; 81:557-66.
GM Crops
67. Dallot S, Quiot-Douine L, Saenz P, Cervera MT, García JA, Quiot JB. Identification of Plum pox virus determinants implicated in specific interactions with different Prunus spp. Phytopathol 2001; 91:159-64. 68. Riechmann JL, Cervera MT, García JA. Processing of the Plum pox virus polyprotein at the P3-6K1 junction is not required for virus viability. J Gen Virol 1995; 76:951-6. 69. Wei T, Zhang C, Hong J, Xiong R, Kasschau KD, Zhou X, et al. Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein P3N-PIPO. PLoS Pathog 2010; 6:1000962; DOI:10.1371/journal.ppat.1000962. 70. Wen RH, Hajimorad MR. Mutational analysis of the putative pipo ofsoybean mosaic virus suggests disruption of PIPO protein impedes movement. Virology 2010; 400:1-7. 71. Dolja VV, Haldeman R, Robertson NL, Dougherty WG, Carrington JC. Distinct functions of capsid protein in assembly and movement of tobacco etch Potyvirus in plants. EMBO J 1994; 13:1482-91. 72. Dolja VV, Haldeman-Cahill R, Montgomery AE, VandenBosch KA, Carrington JC. Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch Potyvirus. Virology 1995; 207:1007-16. 73. Carrington JC, Jensen PE, Schaad MC. Genetic evidence for an essential role for Potyvirus CI protein in cell-to-cell movement. Plant J 1998; 14:393-400. 74. Laín S, Riechmann JL, García JA. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res 1990; 18:7003-6. 75. Laín S, Martín MT, Riechmann JL, García JA. Novel catalytic activity associated with positive-strand RNA virus infection: Nucleic acid-stimulated ATPase activity of the plum pox Potyvirus helicase like protein. J Virology 1991; 65:1-6. 76. Fernández A, Laín S, García JA. RNA helicase activity of the plum pox Potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain. Nucleic Acids Res 1995; 23:1327-32. 77. Fernández A, Guo HS, Sáenz P, Simón-Buela L, Gómez de Cedrón M, García JA. The motif V of Plum pox Potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication. Nucleic Acids Res 1997; 25:4474-80. 78. Hong Y, Hunt AG. RNA polymerase activity catalyzed by a Potyvirus-encoded RNA-dependent RNA polymerase. Virology 1996; 226:146-51. 79. Puustinen P, Makinen KM. Uridylylation of the Potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg. J Biol Chem 2004; 79:38103-11. 80. Schaad MC, Jensen PE, Carrington JC. Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum targeted viral protein. EMBO J 1997; 16:4049-59. 81. Thivierge K, Cotton S, Dufresne PJ, Mathieu I, Beauchemin C, Ide C, et al. Eukaryotic elongation factor 1A interacts with Turnip mosaic virus RNAdependent RNA polymerase and VPg-Pro in virusinduced vesicles. Virology 2008; 377:216-25. 82. Cotton S, Grangeon R, Thivierge K, Mathieu I, Ide C, Wei T, et al. Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments and are each derived from a single viral genome. J Virol 2009; 83:10460-71. 83. Wei T, Huang TS, McNeil J, Laliberte JF, Hong J, Nelson RS, et al. Sequential recruitment of the endoplasmic reticulum and chloroplasts for plant Potyvirus replication. J Virol 2010; 84:799-809. 84. Varrelmann M, Maiss E. Mutations in the coat protein gene of Plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana. J Gen Virol 2000; 81:567-76.
31
85. Candresse T, Cambra M, Dallot S, Lanneau M, Asensio M, Gorris MT, et al. Comparison of monoclonal antibodies and polymerase chain reaction assay for the typing of isolates belonging to the D and M serotypes of plum pox Potyvirus. Phytopathol 1998; 88:198-204. 86. Glasa M, Palkovics L, Kominek P, Labonne G, Pittnerova S, Kudela O, et al. Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically similar and form a unique PPV subgroup. J Gen Virol 2004; 85:2671-81. 87. Myrta A, Varga A, James D. The complete genome sequences of an El Amar isolate of Plum pox virus (PPV) and its phylogenetic relationship to other PPV strains. Arch Virol 2006; 151:1189-98. 88. Serçe CU, Candresse T, Svanella-Dumas L, Krizbai L, Gazel M, Caglayan K. Further characterization of a new recombinant group of Plum pox virus isolates, PPV-T, found in orchards in the Ankara province of Turkey. Virus Res 2009; 142:121-6. 89. James D, Varga A. Nucleotide sequence analysis of Plum pox virus isolate W3174: evidence of a new strain. Virus Res 2005; 110:143-50. 90. Nemchinov L, Crescenzi A, Hadidi A, Piazzolla P, Verderevskaya T. Present status of the new cherry subgroup of Plum pox virus (PPV-C). In: Hadidi A, Khetarpal RK, Koganezawa H, ed. Plant Virus Disease Control. St. Paul, MN: APS Press, 1998:629-38. 91. Candresse T, Cambra M. Causal agent of sharka disease: historical perspective and current status of Plum pox virus strains. EPPO Bull 2006; 36:239-46. 92. Sanford JC, Johnston SA. The concept of parasitederived resistance. Deriving resistance genes from the parasite’s own genome. J Theoret Biolo 1985; 113:395-405. 93. Whitham S, McCormick S, Baker B. The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc Natl Acad Sci USA 1996; 93:8776-81. 94. Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P. Transgenic plant expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 1993; 366:469-72. 95. Petri C, Burgos L. Transformation of fruit trees. Useful breeding tool or continued future prospect? Trans Res 2005; 14:15-26. 96. Regner F, da Camera Machado A, Laimer da Camera Machado M, Steinkellner H, Mattanovich D, Hanzer V, et al. Coat protein mediated resistance to Plum pox virus in Nicotiana clevelandii and N. benthamiana. Plant Cell Rep 1992; 11:30-3. 97. Ravelonandro M, Monsion M, Teycheney PY, Delbos R, Dunez J. Construction of a chimeric viral gene expressing Plum pox virus coat protein. Gene 1992; 120:167-73. 98. Palkovics L, Wittner A, Balazs E. Pathogen-derived resistance induced by integrating the Plum pox virus coat protein gene into plants of Nicotiana benthamiana. Acta Hort 1995; 386:311-7. 99. Guo HS, Garcia JA. Delayed resistance to Plum pox Potyvirus mediated by a mutated RNA replicase gene: Involvement of a gene-silencing mechanism. Mol Plant Micr Inter 1997; 10:160-70. 100. Guo HS, Cervera MT, Garcia JA. Plum pox Potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations. Gene 1998; 206:263-72. 101. Wittner A, Palkovics L, Balazs E. Nicotiana benthamiana plants transformed with the Plum pox virus helicase gene are resistant to virus infection. Virus Res 1998; 53:97-103. 102. Jacquet C, Ravelonandro M, Bachelier JC, Dunez J. High resistance to Plum pox virus (PPV) in transgenic plants containing modified and truncated form of PPV coat protein gene. Trans Res 1998; 7:29-39. 103. Comes S, Fanigliulo A, Piazzolla P, Crescenzi A. Nicotiana benthamiana plants transgenic for PPV-SWC coat protein are resistant to PPV infection. Plant Protec Science 2002; 38:608-11.
32
104. Barajas D, Tenllado F, Gonzales-Jara P, MartinezGarcia B, Atencio FA, Diaz-Ruiz JR. Resistance to Plum pox virus (PPV) in Nicotiana benthamiana plants trasformed with the PPV HC-Pro silencing suppressor gene. J Plant Pathol 2004; 86:239-48. 105. Laimer da Camara Machado M, da Camara Machado A, Hanzer V, Weiss H, Regner F, Steinkellner H, et al. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum pox virus. Plant Cell Rep 1992; 11:25-9. 106. Ravelonandro M, Monsion M, Delbos R, Dunez J. Variable resistance to Plum pox virus and potato virus Y infection in transgenic plants expressing Plum pox virus coat protein. Plant Science 1993; 91:157-69. 107. Scorza R, Ravelonandro M, Callahan AM, Cordts JM, Fuchs M, Dunez J, et al. Transgenic plum (Prunus domestica L.) express the Plum pox virus coat protein gene. Plant Cell Rep 1994; 14:18-22. 108. Tavert-Roudet G, Ravelonandro M, Bachelier JC, Dunez J. Trasgenic Nicotiana benthamiana plant containing the P1 gene of Plum pox virus are resistant to virus challenge. Eur J Plant Pathol 1998; 104:103-7. 109. Jacquet C, Delecolle B, Raccah B, Lecoq H, Dunez J, Ravelonandro M. Use of modified Plum pox virus coat protein genes developed to limit heteroencapsidationassociated risks in transgenic plants. J Gen Virol 1998; 79:1509-17. 110. Ravelonandro M, Scorza R, Bachelier JC, Labonne G, Levy L, Damsteegt V, et al. Resistance of trasgenic Prunus domestica to Plum pox virus infection. Plant Disease 1997; 81:1231-5. 111. Guo HS, Lopez-Moya JJ, Garcia JA. Mitotic stability of infection-induced resistance to Plum pox Potyvirus associated with transgene silencing and DNA methylation. Mol Plant Micr Inter 1999; 12:103-11. 112. Scorza R, Callahan A, Levy L, Damsteegt V, Webb K, Ravelonandro M. Post-transcriptional gene silencing in ©2 Plum 0 pox virus resistant transgenic European 1 1 L an desB i os c i e nc e.plum containing the plum pox Potyvirus coat protein gene. on otdi s t r i but e. Trans Res D 2001; 1054:1-9. 113. Baulcombe DC. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol 1996; 32:79-88. 114. Baulcombe DC. RNA silencing in plant. Nature 2004; 431:356-63. 115. Voinnet O. Post-transcriptional RNA silencing in plant-microbe interactions: a touch of robustness and versatility. Curr Opin Plant Biol 2008; 11:464-70. 116. Voinnet O. Origin, biogenesis and activity of plant microRNAs. Cell 2009; 136:669-87. 117. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM. Total silencing by intronspliced hairpin RNAs. Nature 2000; 407:319-20. 118. Pandolfini T, Molesini B, Avesani L, Spena A, Polverari A. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to Plum pox virus without preventing local infection. BMC Biotechnol 2003; 3:7. 119. Liu Z, Scorza R, Hily JM, Scott SW, James D. Engineering resistance to multiple Prunus fruit viruses through expression of chimeric hairpins. J Am Soc Hort Sci 2007; 132:407-14. 120. Hily JM, Ravelonandro M, Damsteegt V, Bassett C, Petri C, Liu Z, et al. Plum pox virus coat protein gene intron hairpin RNA construct provides resistance to Plum pox virus in Nicotiana benthamiana and Prunus domestica. J Am Soc Hort Sci 2007; 132:850-8. 121. Wang A, Tian L, Huang TS, Brown DCW, Svircev AM, Stobbs LW, et al. The development of genetic resistance to Plum pox virus in transgenic Nicotiana benthamiana and Prunus domestica. Acta Hort (ISHS) 2009; 839:665-72. 122. Di Nicola-Negri E, Brunetti A, Tavazza M, Ilardi V. Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficent and predictable resistance to the virus. Trans Res 2005; 14:989-94.
GM Crops
123. Di Nicola-Negri E, Tavazza M, Salandri L, Ilardi V. Silencing of Plum pox virus 5'UTR/P1 sequence confers resistance to a wide range of PPV strains. Plant Cell Rep 2010; 29:1435-44. 124. Gil M, Esteban O, Martínez MC, Gorris MT, Peña L, Cambra M, et al. Expression of single-chain variable fragments against structural and non-structural proteins of Plum pox virus in transgenic Nicotiana benthamiana plants to interfere with viral infection. Acta Hort 2004; 657:341-7. 125. Boonrod K, Galetzka D, Nagy PD, Conrad U, Krczal G. Single-chain antibodies against a plant viral RNAdependent RNA polymerase confer virus resistance. Nat Biotechnol 2004; 22:856-62. 126. Hily JM, Scorza R, Webb K, Ravelonandro M. Accumulation of the long class of siRNA is associated with resistance to Plum pox virus in transgenic woody perennial plum tree. Mol Plant Micr Inter 2005; 18:794-9. 127. Malinowski T, Cambra M, Capote N, Zawadzka B, Gorris MT, Scorza R, et al. Field trials of plum clones transformed with the Plum pox virus coat protein (PPVCP) gene. Plant Disease 2006; 90:1012-8. 128. Polak J, Pivalova J, Kundu JK, Jokes M, Scorza R, Ravelonandro M. Behaviour of transgenic Plum pox virus resistant Prunus domestica L. clone C5 grown in the open field under a high and permanent infection pressure of the PPV-Rec strain. J Plant Pathol 2008; 90:33-6. 129. Zagrai I, Capote N, Ravelonandro M, Cambra M, Zagrai L, Scorza R. Plum pox virus silencing of C5 transgenic plums is stable under challenge inoculation with heterologous viruses. J Plant Pathol 2008; 90:63-71. 130. Petri C, Webb K, Hily JM, Dardick C, Scorza R. High transformation efficiency in plum (Prunus domestica L.): A new tool for functional genomics in Prunus spp. Mol Breed 2008; 22:581-91. 131. Tepfer M. Risk assessment of virus-resistant transgenic plants. Annu Rev Phytopathol 2002; 40:467-91. 132. Savenkov EI, Valkonen JPT. Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another Potyvirus. J Gen Virol 2001; 82:2275-8. 133. Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, Lakatos L, et al. EMBO J 2003; 22:633-40. 134. Simón-Mateo C, García JA. miRNA-guided processing impairs Plum pox virus replication but the virus readily evolve to escape this silencing mechanism. J Virol 2006; 80:2429-36. 135. Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, et al. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 2006; 24:1420-8. 136. Qu J, Ye J, Fang R. Artificial miRNA-mediated virus resistance in plants. J Virol 2007; 81:6690-9. 137. Duan CG, Wang CH, Fang RX, Guo HS. Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 2008; 82:11084-95. 138. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003; 21:635-7. 139. Lin S, Wu H, Elena S, Chen K, Niu Q, Ye S, et al. Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing. PLoS Pathog 2009; 5:1000312. 140. Duprat A, Caranta C, Revers F, Menand B, Browning KS, Robaglia C. The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to Potyviruses. Plant J 2002; 32:927-34. 141. Decroocq V, Sicard O, Alamillo JM, Lansac M, Eyquard JP, Garcia JA, et al. Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana. Mol Plant Microbe Interact 2006; 19:541-9.
Volume 2 Issue 1
142. Huang TS, Wei T, Laliberte JF, Wang A. A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection. Plant Physiol 2010; 152:255-66. 143. Gutierrez-Campos R, Torres-Acosta JA, Saucedo-Arias LJ, Gomez-Lim MA. The use of cysteine proteinase inhibitors to engineer resistance against Potyviruses in transgenic tobacco plants. Nat Biotechnol 1999; 17:1223-6. 144. Wen R, Zhang SC, Michaud D, Sanfacon HN. Inhibitory effects of cystatins on proteolytic activities of the Plum pox Potyvirus cysteine proteinases. Virus Res 2004; 105:175-82.
145. Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, et al. Engineering stable cytoplasmic intrabodies with designed specificity. J Mol Biol 2003; 330:323-32. 146. Rudolph C, Schreier PH, Uhrig JF. Peptide-mediated broad-spectrum plant resistance to tospoviruses. Proc Natl Acad Sci USA 2003; 100:4429-34. 147. Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, et al. Criteria for Annotation of Plant MicroRNAs. Plant Cell 2008; 20:3186-90. 148. Mlotshwa S, Pruss GJ, Vance V. Small RNAs in viral infection and host defense. Trends Plant Sci 2000; 13:375-82.
149. Díaz-Pendón JA, Ding SW. Direct and indirect roles of viral suppressors of RNA silencing in pathogenesis. Annual Rev Phytopathol 2008; 46:303-26. 150. Burgyán J. Mechanism of action of viral suppressors of RNA silencing. In: Caranta C, et al., ed. Recent Advances in Plant Virology. Caister Academic Press, 2011:137-47. 151. Roth BM, Pruss GJ, Vance VB. Plant viral suppressors of RNA silencing. Virus Res 2004; 102:97-108.
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
www.landesbioscience.com
GM Crops
33
