Virus resistance in orchids

G Model

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Plant Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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Review

Virus resistance in orchids Kah Wee Koh a , Hsiang-Chia Lu a , Ming-Tsair Chan a,b,∗ a b

Academia Sinica Biotechnology Center in Southern Taiwan, Tainan, Taiwan Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Coat protein Cymbidium mosaic virus Odotoglossum ringspot virus Pathogen-derived resistance Post-transcriptional gene silencing

a b s t r a c t Orchid plants, Phalaenopsis and Dendrobium in particular, are commercially valuable ornamental plants sold worldwide. Unfortunately, orchid plants are highly susceptible to viral infection by Cymbidium mosaic virus (CymMV) and Odotoglossum ringspot virus (ORSV), posing a major threat and serious economic loss to the orchid industry worldwide. A major challenge is to generate an effective method to overcome plant viral infection. With the development of optimized orchid transformation biotechnological techniques and the establishment of concepts of pathogen-derived resistance (PDR), the generation of plants resistant to viral infection has been achieved. The PDR concept involves introducing genes that is(are) derived from the virus into the host plant to induce RNA- or protein-mediated resistance. We here review the fundamental mechanism of the PDR concept, and illustrate its application in protecting against viral infection of orchid plants. © 2014 Published by Elsevier Ireland Ltd.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modes of transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viruses prevalent in orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cymbidium mosaic virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Odotoglossum ringspot virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of orchid viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation in orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Particle bombardment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agrobacterium-mediated transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus-resistance in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogen-derived resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coat protein gene-mediated resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA silencing-mediated resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other viral protein gene-mediated resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressor of RNA silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral suppressor of RNA silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VSRs targeting RNA components of RNA silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VSRs targeting AGO proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VSRs targeting proteins associated with RNA silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VSRs interfering with secondary siRNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VSRs interfering with the epigenetic modification of the viral genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting VSRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogen-derived resistance in orchid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generating multiple resistance via gene-stacking approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: Academia Sinica Biotechnology Center in Southern Taiwan, Tainan 741, Taiwan. Tel.: +886 6 5050020; fax: +886 6 5053352. E-mail addresses: [email protected], [email protected] (M.-T. Chan). http://dx.doi.org/10.1016/j.plantsci.2014.04.015 0168-9452/© 2014 Published by Elsevier Ireland Ltd.

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VIGS-based approach to study plant resistance to virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asymptomatic CymMV as an advantage for loss-of-function study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

mechanical transmission by tools and pots contaminated with the viral-containing plant saps. Good horticultural practices such as weed and insect control, and the sterilization of cutting tools and pots prior to use, together with the identification and eradication of virus-infected plants are essential to prevent further spreading of viruses.

Orchids, members of the family Orchidaceae, are highly evolved monocotyledonous flowering plants, comprising 900 genera and 2500–3500 species [1]. They are one of the largest families to exhibit huge diversities in flower size, shape and colors, and are famous for their longevity and enchantingly beautiful appearance. They are, therefore, economically popular ornamental cut-flowers and potted floricultural crops worldwide. Due to their high economic value, new varieties with specific or improved floral traits and desired characteristics, such as flowering time and prolonged vase-life, are constantly being generated to meet the market demand. During cultivation, orchid plants are highly threatened by many phytopathogens, especially the viruses. Due to their high susceptibilities, the cultivation of resistant orchids has become a big challenge to the industry. To date, at least 30 different viruses have been reported to infect orchids, including Cymbidium mosaic virus (CymMV) [2], Odontoglossum ringspot virus (ORSV) [3], Orchid fleck virus (OFV) [4], Cucumber mosaic virus (CMV) [5], Dendrobium vein necrosis closterovirus (DVNV) [6], and Tomato spotted wilt virus (TSWV) [7]. Although these viruses may infect orchids of a specific species, some of these viruses (e.g. CymMV) may produce different diseases in different species. When orchids are virally infected, they may display severe disease symptoms that affect the quality of the flowers, significant ones being floral and foliar necrosis, color-breaking of flowers causing variation in petal color, size reduction, unpleasant leaf appearances (leaf curl), reduced vigor and stunted growth [8]. These symptoms may vary in orchids and are dependent on factors, such as plant genotype (genus, species, variety), environmental conditions (temperature, humidity), plant management (level of nutrition, abiotic and biotic stresses) and types of virus species and isolates. Plants co-infected with bacteria or fungi may also be factors that contribute to these symptoms. Disease significantly reduces the economic value of orchids. Contradictorily, these ‘visible’ detrimental disease symptoms are good indicators of viral infection, and are easily distinguished from the healthy plants. In some cases, infected orchids are asymptomatic and appear visibly indistinguishable from healthy ones. This may hinder disease containment, as these plants are equally infectious to their neighbors. Since there is no effective measure to prevent CymMV and ORSV infections, the eradication of infected plants will help prevent further widespread of viruses. Therefore, routine virus screenings in conjunction with quick and reliable routine diagnostic protocols are required to resolve this problem. Alternatively, the establishment of an efficient anti-viral approach would provide a better solution.

Viruses prevalent in orchids CymMV and ORSV are two of the most prevalent viruses of orchids, both predominantly present worldwide. They are highly stable viruses that possess similar biological and epidemiological characteristics, and are therefore, commonly co-infected. During single-infection, these viruses may produce symptomatic or asymptomatic diseases in some orchid genera, however plants infected with these two viruses produce severe disease symptoms that are more pronounced than a single-infection [9,10]. Orchids that are affected include Cymbidium, Odontoglossum, Phalaenopsis and Oncidium, and symptoms include mottling, ridging, curling and distortion of flowers and abnormal growth and stunting of plants. Symptom severity during co-infection is attributed to the synergism between ORSV and CymMV. Their co-existence results in enhanced RNA replication, hence causing the viral loads to be highly accumulated in infected plants [11]. This synergism greatly affects the flower quality and quantity, making CymMV and ORSV two economically important viruses in orchid cultivation worldwide that are responsible for significant financial losses in the orchid industry. Both CymMV and ORSV are relatively heat-stable, highly virulent, and cause systemic infections that affect all parts of the orchid [12,13]. They are therefore, abundant in plant saps, and capable of retaining their infectivity for a long time [14]. Thus, these viruses are transmitted mechanically by plant sap-contaminated tools and potting media used for plant manipulation and nursery management practice. Gardeners working on orchids have become the main transmission agents responsible for the continuous spread of viruses from one plant to another by the dissemination of viruses to plants present in other nurseries. Besides mechanical transmission, CymMV and ORSV are transmitted by contaminated pollen [15–17]. Pre-sterilization of seeds before sowing will ensure that the young seedling plants are free from both viruses. Cymbidium mosaic virus CymMV was first reported by Jensen [18]. It infects cultivated orchids, and is therefore, also known as Orchid mosaic virus. It is a species of the genus of Potexvirus of the family of Flexivirida. It is a flexuous rod of approximately 500 nm in length and 15 nm in width [19]. CymMV has a monopartite, positive-sense singlestranded RNA genome of 6.3 kb nucleotides, containing five open reading frames (ORFs) flanked by a capped 5 end and a polyadenylated 3 end [20]. ORF1 encodes a 160-kDa replicase that contains three recognized conserved domains, namely a methyl transferase domain in the N-terminal region, an RNA helicase domain and an RNA-dependent RNA polymerase (RdRp) in the C-terminal region. ORFs 2–4 are three overlapping ORFs, that are referred to as the triple gene block (TGB), and the 26-kDa/13-kDa/10-kDa proteins encoded by TGB, also called the movement protein (MP), are required for cell-to-cell movement [21–23]. TGBp1 also functions

Modes of transmission Understanding how different viruses are transmitted will help in controlling viral infections in the orchid industry. Some orchid viruses (e.g. potyviruses) are transmitted by the seeds of an infected plant. OFV, CMV and several other potyviruses and tospoviruses are transmitted by arthropods, such as mites, and insects, such as aphids and thrips. Elimination of weeds that grow near orchids may help eliminate viral transmission. Viruses that infect orchids systemically (CymMV and ORSV) are spread through Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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Fig. 1. Disease symptoms caused by (A and B) CymMV and (C and D) ORSV in leaves of Phalaenopsis orchid. (A and B) Orchids infected by CymMV cause necrotic symptoms on infected leaves. (C and D) ORSV causes yellow stripes symptoms on infected orchid leaves. (E) CymMV CP-transgenic orchid plants (TS1, TS11 and TS16) infected with CymMV exhibit enhanced resistance to CymMV infection as compared to untransformed plant (WTV). The arrow in WTV depicts the symptoms caused by CymMV infection. [E is reprinted with permission from ‘Liao, LJ et al. (2004). Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium Mosaic Virus in a manifestation of RNA-mediated resistance, 13:229–242’ ©2004, Springer, USA].

as an RNA silencing suppressor [24]. The fifth ORF encodes a 24kDa viral coat protein (CP), necessary for virion assembly (viral encapsidation), and which is also involved in cell-to-cell movement. Orchids infected by CymMV are either asymptomatic or show a variety of symptoms that cause chlorotic or necrotic sunken patch symptoms on infected leaves and flowers (Fig. 1A and B), which may affect flower yield. Due to the worldwide predominance of CymMV, molecular variability of CymMV isolates have been extensively studied. In 2002, Ajjikuttira and colleagues compared the CP of 26 Asian isolates [25], and found 89% and 93% identity at the nucleotide and amino acid levels, respectively, with no distinct region of variability in the sequences. In another study, 85 CP and 37 RdRp genes of CymMV isolated from vanilla isolates and other plant sources had a low genetic diversity [26]. Based on phylogenetic studies, CymMV may be categorized into two subgroups based on their nucleotide variations. Although the isolates differ in nucleotide sequences, no clustering in amino acid sequences were seen, suggesting that nucleotide variations are mostly synonymous substitutions. The genetic diversity of CP genes of 108 CymMV isolates from different geographical locations have been compared with 55 CymMV Korea isolates that infect different genera of orchids [27]. The authors

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noted 75% and 53% CP homology at nucleotide and amino acid levels, respectively, between the two groups of isolates. Similar to those observed by Moles and colleagues (2007), no region of variability was found between the two groups of viruses. Overall, this suggests the absence of a positive correlation between the geographical locations of the CymMV and their sequence identities; the CymMV isolates have low levels of diversity despite being situated at different geographical locations. Further analysis suggested that the C-terminal region of CymMV CP amino acid sequences is more divergent than the N-terminal region. Since the CymMV CP proteins of different isolates show little diversity and are not geographically distinct, it suggests a potential use of the N-terminal region as a transgene target for the generation of CymMV-resistant orchids worldwide. Odotoglossum ringspot virus ORSV, first reported to be a pathogen of Odontoglossum grande [3], is a member of the genus, Tobamovirus, of the family of Virgaviridae. It is a monopartite, single-stranded, positive-sense RNA with four ORFs, encoding a 126-kDa RdRp, a 183-kDa ribosomal read-through protein, a movement protein and a CP. It is a rigid et

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rod-shaped particle of ∼300 nm in length and 18 nm in width. Its genome organization is similar to the common Tobacco mosaic virus (TMV), and is therefore also known as the TMV-orchid strain [3]. It infects over 20 genera of the Orchidaceae and produces a wide diversity of disease symptoms in different genera and species. For example, it induces ringspot symptom in O. grande and diamond mottle in Cymbidium [3]. Other symptoms include color-breaking of the flower petals, steak or stripe mosaic or necrotic spots and yellowing of leaves (Fig. 1C and D) [3,28,29]. In Phalaenopsis and Oncidium, disease symptoms produced by ORSV are not usually obvious, with plants having normal growth and flower structures [19]. However, as mentioned above, enhanced disease symptoms are produced when these orchids are co-infected with ORSV and CymMV [19]. The molecular variability of ORSV isolates is also being extensively studied. In 2002, Ajjikuttira and colleagues [25] compared the CP sequences of 20 Asian isolates, and found 96% and 93% identity at the nucleotide and amino acid levels, respectively. Similar to the CymMV CP, no distinct regions of variability were found in the ORSV CP sequences. CP gene sequences of 48 Korea isolates have been compared with 38 isolates from geographically distinct locations [27]. These authors found 95% and 92% CP homology at nucleotide and amino acid levels, respectively, between the two groups of isolates. As observed by Ajjikuttira and colleagues (2002), no region of variability was detected in either of the two groups. Phylogenetic tree and recombination analyses suggest that the OSRV CP of both groups is highly conserved, and the sequence identities and geographical locations between the two groups are not distinctly correlated. Further analysis of the ORSV CP protein sequences between the two groups of isolates suggests that the Nterminal region is more conserved than the C-terminal region [27]. Since the ORSV CP proteins of different isolates have low diversity, and are not geographically distinct, this suggests a potential use of the N-terminal CP domain as a transgene target for the generation of ORSV-resistant orchids.

procedures are specific and sensitive, sample preparation procedures in some cases can be tedious and time-consuming. Therefore, simpler and user-friendly procedures are constantly being developed to give high specificities and sensitivities in a relatively shorter period. In 2011, Lee and colleagues developed a single-tube onestep reverse-transcription loop-mediated-isothermal amplification (RT-LAMP) procedure that allows the CymMV-specific DNA product with a loop structure to be amplified at a single temperature within one hour [39]. Thus, rapid detection of CymMV target RNA with high sensitivity and specificity became possible. Since the primers are designed based on the conserved regions of the CymMV isolates, this also allows the successful detection of CymMV in various orchids, such as Phalaenopsis, Oncidium, Cymbidium and Dendrobium. With the development of RT-LAMP, an automated chip assay that uses integrated microfluid system to carry out viral RNA purification, RT-LAMP amplification of target RNA and the optical detection of the amplified products in a single chip assay were subsequently developed [40]. They significantly reduced the laborious work required for RNA extraction, and allowed easier screening of large numbers of asymptomatic plants for CymMV infection. Besides RT-PCR, a fiber optical particle plasmon resonance (FOPPR) immunosensor-based on gold nanorods has been developed for label-free detection of orchid viruses [41]. The use of gold nanorods as the sensing material eliminates color interference caused by gold nanospheres. Using this antibody-based system, specific recognition of CymMV and ORSV, as well as the diagnosis between infected and healthy plants, is achieved within 10 min. These newly developed detection procedures will allow rapid screening of orchids without compromising specificity and sensitivity. Transformation in orchids New and improved varieties of orchids are constantly produced to meet consumer demand. The traditional classical breeding method utilizing sexual hybridization and selection to produce new orchid hybrids are usually time-consuming due to the long protracted selection period. The successful manipulation of specific floral traits to generate flowers with desirable characteristics, e.g. virus resistance and early flowering time are limited using traditional methods. These have become the major hurdle to the faster generation of new orchid varieties. Fortunately, with the advent of molecular biology in the last two decades, the rapid production and selection of transgenic orchids with genetically modified traits have become possible. The development of gene transfer technology allowing the genetic engineering of specific genes with particular floral traits has overcome the limitations of traditional breeding methods, and the selection and regeneration of orchids with desired traits may be accomplished with high success rate. Currently, transformation methods available for the transfer of exogenous genes into orchid tissues include particle bombardment, Agrobacterium-mediated transformation, electrophoresis of orchid protoplast/protocorms, seed imbibition and transformation by pollen tube pathway [42]. Of these, particle bombardment is the preferred method, followed by Agrobacterium-mediated transformation [43].

Detection of orchid viruses CymMV and ORSV are two of the most prevalent and economically important viruses to infect cultivated orchids, and are responsible for the significant financial losses globally in the orchid industry [30]. As there is no complete cure for viral infection, and some of the infected plants may have asymptomatic diseases, the screening of healthy plants for viral infection, together with the identification of types of viruses infecting the orchids, are crucial for virus-containment and the prevention of transmission. Therefore, the establishment of simple diagnostic procedures with high specificity and sensitivity are required for the control of plant viral diseases. Over the past two decades, many laboratory diagnostic procedures have been developed, targeting the protein and/or the nucleic acid components of the viruses. In 1994, enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of Dendrobium orchids infected with CymMV and ORSV [31]. Subsequently, immunocapture-PCR (IC-PCR) was developed for the diagnosis of both the viral nucleic acids and capsid proteins of CymMV and ORSV [32]. Other nucleic acid-biased detection procedures include reverse transcription-polymerase chain reaction (RT-PCR) [33], digoxigenin (DIG)-labeled cRNA probes [34] real-time RT-PCR [35] and molecular beacon hybridization [36]. For protein-based and viral capsid protein detection, immuno-capillary zone electrophoresis (I-CZE) [8], liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry [37], quartz crystal microbalance (QCM) immunosensors [38] and electron microscopy [19] have been developed. Although some of these Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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Particle bombardment Particle bombardment was first described by Klein and colleague in 1987, whereby DNA is first coated onto metal microcarriers, which are subsequently driven into the plant cells by gas acceleration using a particle gun transformation [44]. It is commonly used in orchid transformation due to its high transformation efficiency.

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Agrobacterium-mediated transformation

(SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) had synergistically resulted in a serious disease in the sweet potato plant. Partial sequences of the CP genes encoded by the four viruses are being used as sources to induce PDR in the transgenic plant [66]. Although viruses are detected in the transgenic potato plants challenged with the viruses, they have delayed or mild disease symptoms compared to non-transgenic plants. Therefore, the introduction of multiple CPs may confer plant resistance to multiple viruses.

Agrobacterium tumefaciens, a Gram-negative bacterium that possesses tumorigenic property, is capable of transferring DNA between itself and plants [45,46]. It is widely used as an important genetic engineering tool for gene transformation in plants. Application of Agrobacterium as a vector for plant transformation has provided many opportunities for genetic engineering in the field of plant biotechnology. With the use today of Agrobacteriummediated transformation, the generation of transgenic plants that express a wide variety of foreign (exogenous) genes has become possible with ease and high efficiency. To date, Agrobacteriummediated gene transformation has been successfully carried in orchid genera, including Oncidium [47], Phalaenopsis [48,49], Dendrobium [50] and Cymbidium [51].

Coat protein gene-mediated resistance Application of the PDR concept to generate TMV-resistant transgenic plant started in 1986 [54]. TMV is a positive-sense single-stranded RNA virus that enters the host cell by mechanical inoculation. Once it has entered the host cell, it becomes uncoated to release its genetic material into the cytosol. With the help of the host machinery, it replicates its genome to produce multiple copies of mRNAs that are translated into coat proteins, RdRp and movement proteins. The RdRp of the virus is involved in its replication. Together with the coat proteins, the RNA genomes are spontaneously assembled into infectious virions that then infect neighboring cells. It is hypothesized that high expression of viral CP protein in the transgenic plant will result in spontaneous assembly of the TMV genome that is released into the cytosol of the host cell. Therefore, the TMV genome becomes unavailable for replication during the early event of viral infection (Fig. 2A) [67,68]. Unfortunately, the expression stages of TMV CP proteins in the transgenic plants are not positively correlated with the intensity of resistance to viral infection. In some transgenic plants expressing low or undetectable levels of CP proteins, high levels of resistance to TMV infections are observed [69]. In another study, tobacco plants expressing an untranslatable CP-encoding gene of Tobacco etch virus (TEV) are resistant to TEV infection [70]. The authors also demonstrated that the RNA segment of the virus genome is responsible for the conferred resistance, hence supporting the presence of an RNA-mediated resistance against the respective virus, however, the mechanism remains poorly understood.

Virus-resistance in plants Pathogen-derived resistance Transgenic plants expressing pathogen-associated gene may be protected against infection by this pathogen. This is achieved through the intervention of viral development or replication, called Pathogen-Derived Resistance (PDR) [52]. PDR of viruses can be divided into two categories: (1) RNA-mediated resistance that involves the transformation of a partial sequence of the virus genome into the plant, and (2) protein-mediated resistance that involves the transformation of the viral full-length proteinencoding gene into the plant [53]. Protein-mediated resistance generally offers a broader range of resistance to the related viruses, but is only effective against a low level of inoculum. RNA-mediated resistance, on the other hand, occurs through a gene silencing mechanism. Although resistance is highly species-specific, it is effective against a high level of inoculum. Application of the PDR concept for antiviral resistance in transgenic plant was first demonstrated in 1986, where transgenic Nicotiana tabacum cv. Xanthi and cv. Samsun expressing the coat protein (CP)-encoding gene of TMV became resistant to TMV infection [54]. These TMV CP transgenic plants are protected against TMV infection to different extents, with some plants showing no or delayed disease symptom development. Following the success of CP-mediated resistance to TMV infection, transgenic tomato plants expressing TMV CP are also protected against infection by various TMV strains [55,56]. With the discovery and development of PDR, this approach has become widely applied to many economically important crop plants, including potato, pea, pepper, papaya, wheat and lettuce, with the hope of generating virus-resistance, with dramatically reduced levels of virus-induced diseases [57–62]. To date, transgenic papaya plants expressing the CP-encoding gene of Papaya ringspot virus (PRSV) have been commercialized, conferring the plant great resistance to PRSV infection in the field [60]. White clover plants expressing the CP-encoding gene of Alfalfa mosaic virus (AMV) also have strong resistance to AMV infection in both the greenhouse and field [63]. Besides generating transgenic plants resistant to a single virus infection, attempts to generate transgenic plants that are simultaneously protected against several viruses have also been investigated [57]. Potato plant that co-expresses the CP-encoding genes of the Potato virus X (PVX) and Potato virus Y (PVY) are simultaneously protected against PVX and PVY infections [64]. Subsequently, transgenic squash plants that are resistant to three viruses, Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and CMV, are generated by introducing the CPencoding genes of these three viruses into the same plant [65]. More recently in South Africa, the Sweet potato feathery mottle virus Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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RNA silencing-mediated resistance The RNA silencing phenomenon is one of the most important findings in the last two decades. In 1990, one group discovered such a phenomenon when they transformed a flowering pigmentation gene, chalcone synthase (CHS), into Petunia [71,72]. Instead of exhibiting colored flowers, some of these transgenic plants produced partially or totally white flowers. Based on RNA protection analysis, the mRNA expression levels of both the endogenous and exogenous CHS mRNA transcripts during the developmental stage were unaltered in both the white and wild-type flowers. However, the CHS mRNA levels of the white-flowered transgenic plants were significantly reduced in the cytosol compared to the wild-type. This suggests that the foreign gene (exogene) induced the degradation of both the introduced and endogenous homology genes at the mRNA level in the cytosol. This phenomenon of gene co-suppression is also known as post-transcriptional gene silencing (PTGS) [73]. Subsequently, Lindbo and colleagues (1993) demonstrated that RNA-mediated resistance is associated with the co-suppression phenomenon [69]. Some of their transgenic tobacco plants expressing TEV CP were initially susceptible to TEV infections. However, several weeks after infection, newly developed stems and leaves had symptomless phenotypes, and were viral-free. The mRNA expression level of the transgene present in the recovered transgenic plant tissues was significantly lower than the non-inoculated transgenic plant tissues. However, the transcription rates of the transgene were similar in both tissues. The recovered tissues had TEV-specific resistance. These authors proposed that the recovered et

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Fig. 2. Mechanisms underlying (A) protein-mediated and (B) RNA-mediated resistances to viral infection. (A) High expression of viral CP in the transgenic plant will result in spontaneous viral assembly and/or become unavailable for replication of virus genome. The dashed arrows with crosses indicate the repression of virus multiplication. (B) The viral dsRNA serves as template to produce siRNA by Dicer cleavage. The viral associated siRNA is incorporated into the RNA-induced silencing complex (RISC) and then recruited to degrade the virus genome.

phenomenon is mediated by sequence-specific RNA degradation induced by the transgene similar to the co-suppression phenomenon. Many other studies have confirmed this theory; the mechanism of co-suppression, also known as PTGS, and expanded research in this field. Post-transcriptional gene silencing (PTGS), also known as RNA interference (RNAi), is an evolutionarily conserved process found in most eukaryotic organisms ranging from fission yeast to man (Fig. 2B). Two classes of small RNAs (sRNAs) – the small interfering RNAs (siRNAs) and microRNAs (miRNAs) of approximately 21–30 nucleotides – are the key players involved in RNA-mediated gene silencing. These non-coding sRNAs destabilize mRNA or/and terminate protein translation by binding specifically to their complementary mRNA targets [73–75]. The precursor RNAs, double-stranded RNAs (dsRNAs) or hairpin RNAs are the initial molecules being processed during the biogenesis of sRNAs. RNase III enzyme, Dicer, is responsible for the cleavage of the precursor RNAs to generate sRNAs that are 21–30 nucleotides in length. In order to possess a gene regulatory function, a process termed

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“loading” is mediated, whereby the generated sRNA is incorporated into a large multiple protein complex, the RNA-induced silencing complex (RISC) [76]. Typically, only one strand of sRNA is associated with one RISC, and this complex is then recruited to the targeted site of the mRNA. Once the RISC is bound to its targeted mRNA, an important effector protein of RISC, termed argonaute (AGO), degrades mRNA and/or inhibits protein translation, resulting in post-transcriptional gene silencing [77]. To date, RNAi seems to have important functions in diverse biological processes, including development, homeostasis, innate immunity, stem-cell regeneration and oncogenesis [78]. Typically, the function of siRNA may be regarded as a virus defense system, since the exogenous viral replication intermediates or endogenous retro-transposon transcripts of the virus are potential RNA precursors of siRNAs [79]. While the precursors of miRNAs are generated as an independent transcriptional unit encoded in the genome, miRNAs are usually involved in transcriptional silencing of the endogenous mRNAs [80]. The biogenesis and functional pathways of different RNAi are diverse among organisms.

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Other viral protein gene-mediated resistance Replication-associated proteins. Besides CP, other components of the virus are potential sources for generating PDR-induced resistance to viral infection. Transgenic plants expressing PVX, PVY or CMV replicase are protected against viral diseases in a protein-mediated manner. Transgenic plants that express mutated replicase with the GDD domain being deleted, PVY replicase with the Nlb domain deleted, or CMV replicase containing the untranslated 2a domain, are not protected against the respective viral infections [81,82]. However, transgenic plants expressing AMV P2 replicase with a mutation in the GDD motif is more resistant to the virus compared those expressing the full-length P2 replicase protein [83]. Golemboski and colleagues (1990) found that transgenic tobacco plant expressing a 54-kDa RdRp of TMV can inhibit viral replication when infected with either TMV U1 virions or TMV U1 infectious RNA [84]. The copy number of the 54-kDa transgene expressed did not influence the level of resistance in these plants. However, when the transgenic plants expressed the truncated 54kDa protein comprising only 20% of the gene segment, it can inhibit viral replication [85]. This suggests that the full-length 54-kDa protein is required to confer plant protection against the respective viral infection. In the case of other tobamovirus, such as the Pepper mild mottle virus (PMMoV), expression of either the truncated 54-kDa protein comprising 30% of the gene segment or the fulllength protein are equally capable of conferring protection of the pepper plant against the respective viral infection [86,87]. Based on these observations, the efficiencies of protein-mediated and RNAmediated protection against viral infection is dependent on the type of viruses.

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process termed virus-induced gene silencing (VIGS) [94,95]. When the host anti-viral surveillance system recognizes a foreign viral RNA, the RNA silencing machinery is activated to target and process the virus-derived dsRNA, which is either derived from pathogen replication or amplified by the host in a RdRp-dependent manner, into vsiRNA (virus-derived siRNAs). These vsiRNAs are then recruited to the host RISC to target and destroy viral RNAs. This in turn, hinders the accumulation of viral RNA and affects the infection process [96]. In cases where plant mRNA has sequence homology with the viral genome, both the viral and plant transcripts will be targeted for destruction [97,98]. Similarly, when plants are transformed to express transgene-induced PTGS, the exogenous foreign transgenic sequence is recognized and amplified by the plant-encoding RdRp, into dsRNA, which serves as the downstream substrate to trigger RNA silencing [99,100]. The resulting PTGS can target against transcripts of the transgenes and any homologous pathogen endogenous genes for degradation, such that the corresponding gene products are greatly reduced [101]. In addition, PTGS may confer cross-protection against infection with a second virus in a nucleotide sequence-specific manner [96]. Besides protecting infected cells against viral infection, PTGS are capable of moving in-between cells via plasmodesmata, and migrate systemically over long distance through the vascular system to direct sequencespecific degradation of target RNAs at a distal site [102,103]. This movement of VIGS signal in advance of the infection front may potentiate systemic RNA sequence-specific virus resistance in the non-infected tissues and consequently, delay the spread of virus. Therefore, the abundant expression of dsRNAs to trigger efficient RNA silencing becomes crucial for effective resistance. Viral suppressor of RNA silencing

Movement-associated protein. Transgenic plants expressing dysfunctional movement proteins (dMP) of virus have a broader range of resistance against viral infection compared to those expressing either the CP or replicase protein. Those expressing dysfunctional TMV dMP (P30) protein can inhibit the local and/or long distance movement of TMV, delaying viral accumulation and the development of symptoms [88]. They are also resistant to taxonomically distant viruses, including Tobacco rattle virus (tobravirus), Peanut chlorotic streak virus (caulimovirus) and Tobacco ringspot virus (nepovirus) by disrupting systemic movement, but not cell-to-cell movement, of the viruses [89]. Unlike TMV with a single movement protein, the potexvirus possesses three movement proteins, the TGB proteins. Transgenic plants expressing a dysfunctional TGB protein, such as the 13-kDa protein of White clover mosaic virus (WCMV) or 12-kDa protein of PVX, are resistant to other TGB-containing viruses, but not to TMV and PVY viruses, respectively [90,91]. Compared to a TMV dMP transgenic plant that only influences the systemic movement of the virus, transgenic plants expressing dysfunctional TGB can inhibit both cell-to-cell and systemic movement of the viruses. In addition to RNA virus, transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement protein of a geminivirus Bean dwarf mosaic virus (BDMV), which is a DNA virus, also are delayed in the development of disease symptoms to Tomato mottle virus (ToMoV) infection [92].

Plant viruses have evolved several strategies to counteract the anti-viral defense mechanism of the host. One of the primary counter-defense measures involves the encoding of a protein called viral suppressor of RNA silencing (VSR), which are encoded in the genomes of both RNA and DNA viruses [95,104–106]. VSR is capable of suppressing the RNA silencing at different steps, either by binding siRNA duplex, or by directly interacting with key components of RNA-silencing pathways. Some of these VSR may have combinatorial functions that allow multilevel suppression [107]. The molecular mechanisms of VSR include targeting proteins/siRNAs associated with gene silencing components (siRNA, AGO and DCL proteins) and disrupting the secondary siRNA amplification or epigenetic modification of viral genome [108]. These molecular mechanisms are briefly described below: VSRs targeting RNA components of RNA silencing The VSRs of viruses may act on dsRNAs or siRNAs to inhibit the production of siRNAs or the incorporation of siRNAs into the RISC, respectively. The B2 protein encoded by Flock house virus (FHV) contains a dsRNA binding domain that binds various lengths of dsRNAs in a dimer form, hence inhibiting their processing into siRNAs [109]. Similarly, the P14 protein encoded by the Pothos latent aureusvirus (PoLV) binds dsRNA to suppress the accumulation of vsiRNA, and at the same time, inhibits the systemic antiviral defense system [110]. The P21 protein of Beet western yellow virus (BWYV) [111] and P19 proteins of Carnation Italian ringspot virus (CIRV) and Tomato bushy stunt virus (TBSV) bind specifically to siRNA [112–114], hence inhibiting the incorporation of siRNA or miRNAs into the active RISC [115–117]. Besides targeting the RISC assembly system at the local infection site, P19 protein of Cymbidium ringspot virus (CymRSV) promotes systemic virus infection by sequestering the vsiRNAs, thus preventing the RNA silencing signals from spreading out of the vascular bundles [118].

Suppressor of RNA silencing As described previously, RNA silencing is a nucleotide sequencespecific process that induces mRNA degradation or translation inhibition at the post-transcriptional levels. It is also involved in epigenetic modification at the transcriptional level, and is dependent on RNA-directed DNA methylation (RdDM) [93]. Besides the involvement in diverse biological processes, RNA silencing also functions as a natural anti-viral defense mechanism, via a Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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VSRs targeting AGO proteins AGO is a key player of RNA silencing, responsible for the degradation of target mRNA and repression of protein translation [119]. VSRs may act on AGO, either by inhibiting its slicer function or inducing its degradation. The 2b protein of CMV interacts directly with AGO1 and AGO4 to inhibit its slicer activity, and competes with AGO4 for binding to siRNA, hence suppressing the DNA methylation function of AGO4 [120]. The P0 (F-box protein) of BWYV, Cucurbit aphid-borne yellows virus (CABYV) and Potato leafroll virus (PLRV) destabilizes AGO1 protein for degradation [121–123]. Another protein, P25 of PVX, which is a movement protein, is capable of interacting with multiple members of the AGO family and causes the degradation of AGO1 in a proteasomedependent manner [124]. Although this VSR fails to induce gene silencing locally, P25 suppresses the propagation of RNA silencing signals out of the infected cells, hence allowing the systemic movement of the viruses [24,125]. This suggests that P25 protein possess combinatorial functions that allow multilevel suppression of RNA silencing.

presence of VSRs may compromise such gene regulation. For example, soybean seeds exhibiting dark, irregular streaks (molting) is caused by the VSRs of the Potyviridae and Cucumoviridae families that suppresses the RNA silencing of CHS gene involved in pigment synthesis [144,145]. Therefore, some of the symptoms displayed during viral infection are caused by VSRs acting on the miRNAs regulating host gene expression. Targeting VSRs Through the understanding of how VSRs act to suppress RNA silencing, it may be used as potential target for the engineering of viral-resistant plants. To date, several attempts have been made to target the VSRs for antiviral approach. In a study by Niu and colleagues [146], transgenic Arabidopsis expressing artificialmiRNAs (ami-RNAs) targeting the VSRs, P69 protein of Turnip yellow mosaic virus (TYMV) and HC-Pro of Turnip mosaic virus (TuMV), exhibited specific resistances to both viruses. Transgenic N. tobacum expressing ami-RNA targeting two VSRs, 2b protein of CMV and TGBp1/P25 of PVX, also exhibited resistances to both viruses [147,148]. Although these ami-RNA mediated resistances confer protection against viral infection, plants exhibited different levels of resistance [148]. Several factors may account for such observations: (1) the ami-RNA target site is not the optimal RISC-accessible site for effective destruction of the VSRs; (2) the positional effects and secondary structures of the viral genome may block RISC accessibility to the target site; (3) the viruses have evolved natural mutation to escape the miRNA-targeting pressure of the host [149]; and (4) the presence of multiple genomes that results in the replication of non-target genomes [150,151]. In a study to overcome the resistance caused by the factors mentioned above, the 3 UTR of CMV, which is functionally essential for CMV replication and highly conserved in different strains, was used to determine the RISC-accessible cleavage hotspots via molecular methods [151]. When transgenic Arabidopsis and tobacco plants were transformed to express ami-RNAs targeting the RISC-accessible hotspots, but not RISC-inaccessible regions, they exhibited high resistances to two strains of CMV. Therefore, the use of ami-RNAs to target the conserved RISC-accessible hotspots of VSRs offer a higher and broader spectrum resistance than those targeting VSR sequences of RNA viruses with multiple genomes. The use of VSRs-targeting ami-RNAs, in conjunction with CP-mediated resistance may confer high levels of protection against the corresponding viral infection.

VSRs targeting proteins associated with RNA silencing The P6 protein of Cauliflower mosaic virus (CaMV) is capable of silencing transgene and prevents the accumulation of endogenous trans-acting siRNAs [126]. When P6 protein is localized in the nuclear, it interacts and suppresses the dsRNA-binding protein DRB4, involved in the production of DCL4-dependent 21-nt siRNA derived from exogenous transgene or viral RNAs [127]. Therefore, P6 protein acts as a VSR that suppresses the production and accumulation of siRNAs, and inhibits both local and systemic RNA silencing. Similarly, the potyviral helper component proteinase (HC-Pro) inhibits the function of DCL involved in the degradation of viral-derived dsRNAs into siRNA, hence enhancing the replication of many unrelated viruses [128,129]. VSRs interfering with secondary siRNA amplification RdRps, in particular RdRp1 and RdRp6, are capable of recognizing viral RNAs and generate secondary vsiRNA for RNA silencing [130–132]. VSRs may act on RdRps to inhibit the synthesis of vsiRNAs. The 2b protein of CMV [133] and V2 protein of Tomato yellow curl leaf Gemini-virus-Isreal (TYLCV-ls) [134–136], suppress the functional role of RdRp6, hence inhibiting the amplification of RdRp6-dependent vsiRNA, which is essential for antiviral defense. Besides amplifying vsiRNAs, RdRp6 is also involved in the migrating of RNA silencing signals between cells [137]. Therefore, the inactivation of RdRp6 by VSRs may promote systemic virus infection.

Pathogen-derived resistance in orchid

VSRs interfering with the epigenetic modification of the viral genome DNA methylation plays an important role in epigenetic modification of gene expression and DNA viral repression. Besides regulating PTGS, sRNA is also involved in transcriptional gene silencing (TGS) via DNA methylation. One good example is the methylation of the genome of ssDNA geminivirus during infection such that the virus becomes defective in replication and infection [138–140]. Viruses of the geminivirus family have evolved to possess suppressors that interfere with the biochemical pathway of DNA methylation [141]. The AL2 protein of Tomato golden mosaic virus (TGMV) and L2 protein of Beet curly top virus (BCTV) can interact and inactivate adenosine kinase (ADK) required to catalyze adenosine into AMP [141,142]. This AMP substrate is required for sustaining the S-adenosyl methionine cycle for genome-wide cytosine methylation [143]. Therefore, the inactivation of ADK by AL2 and L2 allows the reversion of established TGS and prevents the epigenetic modification of the viral genome. Besides functioning as an anti-viral defense mechanism, RNA silencing is also important in regulating host gene expression. The Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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Although the routine screening of healthy plants and the eradication of viral-infected plants, in conjunction with good horticultural practices, may aid in viral containment, generation of orchids with enhanced resistance to viral infection should provide an alternative solution to the infection problem. With advances in plant transformation, the establishment of the pathogen-derived resistance (PDR) concept has become established and is widely used in generating transgenic plants that are resistant to pathogens, such as viruses and bacteria [52]. High CP sequence homologies are found in both CymMV and ORSV isolates, which suggests the feasibility of using CP as a transgene to produce transgenic orchids resistant to these viruses, regardless of their geographic origin. Based on the studies by Ajjikuttira and colleagues (2002), the CP-encoding regions of amino acid 107–121 and 18–40 of CymMV and ORSV, respectively, are highly conserved among all the isolates, which are therefore the potential regions for the production of transgenic orchids resistant to their respective viruses [25]. With the successful development et

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of the PDR concept and optimized transformation protocols, generation of orchids resistant to CymMV and/or ORSV infections has been attempted using the CP gene-mediated mechanism. The CP gene-mediated resistance concept has been applied to orchid plants to generate Phalaenopsis and Dendrobium that are resistant to CymMV infection. In 2004, Chan’s laboratory successfully transformed the CymMV CP-encoding gene into the Phalaenopsis protocorm-like bodies (PLBs) by particle bombardment [152]. Four putative transgenic lines with rapid growth rates, and harboring a CP-encoding gene at different insertion sites were examined for their resistance to CymMV infection. Although these CymMV CP transgenic plants are transformed to express the CP-encoding gene, low CP mRNA transcript levels with no CP protein expression were detected. Upon being challenged with CymMV virions for 30 days, CymMV CP protein accumulated in the wild-type Phalaenopsis plants, but not in any of the four CymMV CP-transgenic plants, which signifies that the wild-type, but not the transgenic Phalaenopsis plants were infected with CymMV, hence suggesting that the CymMV CP transgenic plants had become resistant to CymMV infection (Fig. 1E). Besides observing CymMV CP transcripts, small interfering RNAs (siRNAs) of CP-encoding gene are detected in the transgenic plants. Overall this suggests that the CymMV CP transcripts expressed are immediately degraded into siRNAs, which act downstream as gene silencers to inhibit the translation of the CP protein. This accounts for the low levels of CP mRNA transcripts and absence of CP proteins in the transgenic plants. Since CP is a major component of the virus, inhibition of CP expression by post-transcriptional gene silencing may result in the failure to package the replicated genome materials into infectious virions. This could explain the inability of the CymMV to multiply and infect neighboring cells. These results suggest that the expression of CymMV CP confers protection against CymMV infection in a RNA-mediated but not protein-mediated manner of intransgenic Phalaenopsis plants. In 2005, Chang and colleagues also successfully generated CymMV CP transgenic Dendrobium plants using biolistic transformation [50]. These transgenic Dendrobium plants challenged with CymMV virions have extremely low levels of virus accumulation and show very mild disease symptoms compared to the wild-type. With the application of the PDR concept, orchid plants that are resistant to CymMV infections can be produced, which may help to solve the pathogen threat posed by the CymMV. Besides the CymMV CP, attempts to generate orchids resistant to ORSV infection have also been made, but with limited success. In 2006, Chen and colleagues [153] transformed the rhizomes of Cymbidium niveo-marginatum with the OSRVCP-encoding gene derived from OSRV-infected Epidendrum. Although the transgenic plants express OSRV CP transcripts in the shoot, their resistance to OSRV infection has not been mentioned. Similarly, Fan found that both the CP-encoding gene of CymMV and OSRV can be transformed into Phalaenopsis amabilis by particle bombardment, thereby expressing the CymMV-ORSV CP fusion protein gene [154]. Although the CP fusion transcripts and proteins are detectable in the transgenic plant, resistance to both CymMV and ORSV infections was not reported. Despite several attempts to generate plants that are resistant to either ORSV or both CymMV and ORSV, a transgenic orchid resistant to ORSV infection has not been reported. This may be due to the failure of the host cells to generate siRNA that target the RNA silencing machinery, resulting in the failure to inhibit viral replication. Therefore, the generation of transgenic plants expressing the siRNA sequences of ORSV CP may be an alternative strategy for further study. Yu and Wong (1998) found that in vitro synthesis of full-length DNA of ORSV genome under a bacteriophage T7 RNA polymerase promoter results in the generation of capped-RNA transcripts that are highly infectious when mechanically inoculated onto seedlings Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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of Nicotiana benthamiana and Oncidium Gower Ramsey [155]. One representative clone, pOT2, had a similar disease phenotype as the parental viral RNA. pOT2 was further mutated to be deficient in CP expression (pOCP1) to determine the role of CP in viral replication and movement. In Chenopodium amaranticolor, the in vitro transcript from pOCP1 elicited necrotic local lesions at five days post inoculation and symptoms were similar to those produced by the parental ORSV-S1. This suggests that the transcripts from pOCP1 are infectious and dispensable for RNA replication in C. amaranticolor. However, in the case of N. benthamiana, in vitro transcripts of pOCP1 cannot cause systemic symptoms. The instability of the pOCP1 transcript is due to the lack of protection by the CP. When the mobility of pOCP1 virus is assayed, cell-to-cell movement occurs in the inoculated leaves of C. amaranticolor, similar to that of the parental virus, and the systemic movement of the pOCP1 virus is possible, but the speed is compromised. The authors concluded that, in the absence of CP, ORSV remains infectious and mobile, thereby suggesting that the CP of ORSV is dispensable for replication and movement. Since the OSRV CP transgenic plant is generated to target CP expression of OSRV, this may explain the failure of the OSRV CP-transgenic orchid plant to become resistant to OSRV infection. The use of other viral components may be more appropriate for the generation of ORSV resistance. Generating multiple resistance via gene-stacking approach Since plants can be infected with a wide range of phytopathogens, it would be ideal to generate transgenic plants resistant to more than one pathogen. The use of gene stacking that allows the transfer more than one trait may be the right approach for the development of multiple resistances. Using gene-stacking approach, Chan and colleagues (2005) first transformed Phalaenopsis with CymMV CP cDNA by particle bombardment of protocorms [156]. The putative PLB transformants that survived antibiotic selection and were resistant to CymMV via PTGS were subsequently transformed with an anti-microbial sweet pepper ferredoxin-like protein cDNA (pflp) by agro-infiltration. Using this gene stacking strategy, double-transformed PLBs generated were proven to be resistant to both CymMV and soft-rot disease-producing Pectobacterium carotovora (formally known as Erwinia cartovora). Since the application of gene stacking allows the generation of plants that are resistant to two different phytopathogens this transformation strategy may be used to generate plants that are resistant to more than one type of viruses, e.g. resistance to both CymMV and ORSV infection. VIGS-based approach to study plant resistance to virus When a plant is infected with a biotrophic pathogen, such as virus, it expresses salicylic acid, triggering a salicylic acid (SA)related plant defense response [157]. The accumulation of SA in turn triggers endogenous signaling that induces the expression of pathogenesis-related (PR) genes [158–160]. SA also elicits a systemic acquired resistance (SAR) response that protects the plant against a broad spectrum of pathogens distal to the infection site. Genes involved in SA-related plant defense response have been extensively studied recently, and genes such as NPR1 have been identified to be involved in the transduction of SA signal for PR gene expression [158,161,162]. NPR1 is a conserved central positive regulator of SA signaling, functioning in its reduced monomeric form to regulate the expression of defense-related genes through the action of TGA and WRKY transcription factors [163]. Using VIGS to elucidate the transcription factors involved in virus-induced plant defense response in P. aphrodite, a PhaTF15 gene located upstream of PhaNPR1 has et

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been identified as involved in PhaNRP1 gene regulation [164]. The knock-down of PhaTF15 by VIGS results in a reduced expression of PhaNRP1, accompanied by an increase in CymMV accumulation. This suggests the possible use of VIGS to look for virus-induced PR genes or TFs important in plant resistance against viral infection. The overexpression of PR genes crucial for plant resistance may protect the plants against viral infection. The interaction between the host cell-surface receptor and viral surface structure, termed tropism, is required for viral entry into the host cells. To date, the tropisms of CymMV and ORSV in plants have not been identified. Using VIGS approach, the cell surface receptors of orchids may be knocked-down to elucidate possible receptors responsible for viral tropism. Once identified, the mutation of these receptors may inhibit viral entry into a plant.

to vary. Using the gene-stacking approach, the introduction of a CP together with a non-CP-based PDR of the same virus (CymMV and ORSV) may confer the orchids more resistance to the respective viral infection. More studies are required to shed further light on the appropriate use of RNA- and protein-mediated resistance to optimize protection against viral infections. Acknowledgements We thank the Plant Transformation Core Facility in Biotechnology Center in Southern Taiwan, Academia Sinica for technical support in identification of some of the overexpressing transgenic orchid lines. We thank Ms. Miranda Loney for helpful suggestion and discussion. This work was supported by the Academia Sinica, Taiwan.

Asymptomatic CymMV as an advantage for loss-of-function study

References

Phalaenopsis orchid usually requires 2–3 years to transit from a vegetative to reproductive phase. It is therefore time-consuming and tedious to study the functional genomics of the genes. An alternative strategy that uses loss-of-function approach was established by Lu and colleagues (2007) to study the function of the gene. A CymMV-based vector that allows specific silencing of target genes by VIGS was established [165]. Together with the isolated CymMV that is asymptomatic and does not cause any physiological change upon infection in Phalaenopsis, the CymMV-based vector containing the gene to be silenced could be transformed into the plant by agro-infiltration. Using this VIGS approach, the insertion of a small fragment of the “gene-of-interest” into the CymMVbased vector, will result in the sequence-specific degradation of the mRNA transcripts. This allows the specific gene of Phalaenopsis to be knocked-down by the gene-silencing mechanism in less than two months. VIGS is induced at a specific developmental stage of the plant, and the essential genes required for plant development is therefore, unaffected. This system allows high throughput functional analysis of plant genes with fast turn-around times and efficiency, which has helped to overcome the challenges of the functional genomic studies faced in orchid technology. However, the efficacy of VIGS is progressively reduced with time, and is ineffective after eight weeks post-inoculation. With the establishment of VIGS, the functional roles of several genes have been investigated [164,166]. Since CymMV can infect a wide range of Orchidaceae, the use of a CymMV-based vector will allow its application on different orchid genera.

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Conclusion and future directions With advances in orchid transformation techniques, genetic engineering of transgenic orchids for molecular genetics and gene functional studies is no longer a challenge in the field of plant biology. In conjunction with the establishment of the PDR concept, transgenic orchids resistant to phytopathogens, such as CymMV and P. carotovora, can be generated to eliminate infection. These will greatly reduce the financial loss incurred from phytopathogen diseases. Despite the success of using CymMV CP as the PDR for plant resistance, the generation of ORSV-resistant orchid is currently faced with setbacks for unknown reasons. Investigation of OSRV CP dispensability for viral replication and mobility in orchids may provide clues for the failure of plants to confer resistance to OSRV infection. Alternatively, the use of non-CP-based PDR, such as ORSV replicase, RdRps, dysfunctional movement proteins and ami-RNA targeting VSRs, may help to solve the problem of generating OSRV-resistant orchids. Although CymMV CP may confer plant resistance to CymMV infection, the level of protection seems Please cite this article in press as: http://dx.doi.org/10.1016/j.plantsci.2014.04.015

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