Roles of MicroRNA in Plant Defense and Virus Offense Interaction

Plant Cell Rep (2008) 27:1571–1579 DOI 10.1007/s00299-008-0584-z

REVIEW

Roles of microRNA in plant defense and virus offense interaction Yan-du Lu Æ Qin-hua Gan Æ Xiao-yuan Chi Æ Song Qin

Received: 26 March 2008 / Revised: 24 June 2008 / Accepted: 25 June 2008 / Published online: 15 July 2008 Ó Springer-Verlag 2008

Abstract MicroRNAs (miRNA) that are around 22 nucleotides long non-protein-coding RNAs, play key regulatory roles in plants. Recent research findings show that miRNAs are involved in plant defense and viral offense systems. Advances in understanding the mechanism of miRNA biogenesis and evolution are useful for elucidating the complicated roles they play in viral infection networks. In this paper a brief summary of evolution of plant antivirus defense is given and the function of miRNAs involved in plant-virus competition is highlighted. It is believed that miRNAs have several advantages over homology-dependent and siRNA-mediated gene silencing when they are applied biotechnologically to promote plant anti-virus defense. miRNA-mediated anti-virus pathway is an ancient mechanism with a promising future. However, using miRNAs as a powerful anti-virus tool will be better

Communicated by P. Kumar. Y. Lu YanTai Institute of Coastal Zone Research for Sustainable Development, Chinese Academy of Science, 264003 Yantai, China Y. Lu
X. Chi
S. Qin (&) Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China e-mail: [email protected] Y. Lu
X. Chi Graduate School of the Chinese Academy of Sciences, 100049 Beijing, China Q. Gan Technical Center of Inspection and Quarantine, Shandong Entry-Exit Inspection and Quarantine Bureau, 266001 Qingdao, China

realized only if miRNA genomics and functions in plant viral infection are fully understood. Keywords MicroRNA
Virus
Plant
Viral infection
Co-evolution

Introduction Viruses cause great loss to plants. In the course of evolution, plants have developed complicated mechanisms to resist viral epidemics. One of the actions is gene silencing. Recent works show that microRNAs (miRNAs) are involved in modulating plant viral diseases (Dunoyer et al. 2004; Carmen and Juan 2006). miRNAs are single-stranded RNA molecules of around 22 nucleotides in length (Ambros 2001), and are derived from larger precursors that are transcribed from non-protein-coding RNA (Bartel 2004; Yu and Kumar 2003). An increasing number of miRNAs have been identified and deposited in major miRNA databases (http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml). Among them, 863 are plant miRNAs (Xie et al. 2007). They are involved in plant development, signal transduction, protein degradation, and response to environmental stress and pathogen invasion. Viruses are known to exploit the host nucleic acids as a part of their infection strategy. Granted that miRNA-mediated gene silencing serves as a general defense mechanism against plant viruses, it would not be a surprise that viruses also employ miRNAs to circumvent the defense system. The discovery of miRNAs has opened up a new avenue for understanding gene expression, plant genetic engineering, and plant pathogenesis molecular investigations. This review highlights the roles of miRNAs in virus offense and plant defense. We also discuss the possible use of miRNAs in combating viral infection.

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miRNA biogenesis and evolution The biogenesis mechanisms of miRNAs are different for plants and animals. In plants, polymerase II transcribes miRNAs into primary miRNA transcripts (pri-miRNA). In the nucleus, a ribonuclease III-like nuclease (DICER-LIKE 1, DCL1) then processes the pri-miRNA with the assistance of one or more unknown enzymes. This process yields a precursor miRNA (pre-miRNA) and ultimately a mature miRNA:miRNA* duplex. The duplex is then exported to the cytoplasm, unwound and incorporated into the RISC complex (Bartel 2004). The miRNA then guides the complex to its specific target mRNA (Llave et al. 2002). However, in animals, the final maturation step is mediated in cytosol by Dicer. The history of miRNAs serving as gene regulators dates back to more than 400 million years ago. Chlamydomonas reinhardtii, a unicellular green alga, has been shown to encode miRNAs (Zhao et al. 2007). It is suggested that the miRNA pathway is an ancient mechanism of gene regulation and it occurred prior to the emergence of multicellularity. This also suggests that miRNAs may have a common ancestor in evolution (Zhang et al. 2005). Two Arabidopsis miRNAs are known to be capable of regulating genes in HD-Zip gene family, and they were found conserved in all lineages of land plants, including bryophytes, lycopods, ferns and seed plants (Floyd and Bowman 2004). Greater evolutionary conservation of miRNAs than siRNAs was proposed (Bartel and Bartel 2003). Most miRNAs harbor imperfect homology with their targets. Therefore, miRNAs are once thought not to affect RNA stability, but to inhibit translation by a RISCdependent mechanism. However, recent research indicated that miRNAs can induce degradation of mRNA in both plants and animals (Chendrimada et al. 2007 and Eulalio et al. 2007). miRNAs control gene expression by regulating mRNA stability and translation (Pillai et al. 2006; Meister 2007; Eulalio et al. 2008). Moreover, miRNAs must mediate post-transcriptional gene silencing by more than one mechanism (Eulalio et al. 2007; Dorner et al. 2007). Otherwise, the target sequences would have to co-evolve (Maher et al. 2006). This idea is supported by the finding that class III homeodomain-leucine zipper (HD-Zip) genes, one of the targets of miR166, have conserved miR166 target regions, whereas other regions have lower nucleotide conservation (Floyd and Bowman 2004). No conservation exists between animal and plant miRNAs according to published reports. However, miRNAs are well conserved among distantly related plant species. Computational prediction revealed that many miRNA families were evolutionarily conserved across all major lineages of plants (Zhang et al. 2005, Zhang et al. 2006). This is in agreement with the observation that miR165/166

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is conserved among angiosperms, ferns, lycopods and mosses (Floyd and Bowman 2004). However, the regulation of a given miRNA may not be similar in diverse plant species. Arabidopsis miR159 was found to be regulated by gibberellin (Achard et al. 2004; Wang et al. 2004a, b). In contrast, miR159 expression in rice seedlings remained unaltered in response to gibberellin application (Tsuji et al. 2006). Furthermore, miRNAs sequence conservation may not indicate a conserved function as shown by the fact that ptr-miR473a, ptrmiR478a, and ptr-miR482 play different roles in Populus compared with rice (Lu et al. 2005). The variety of miRNAs must have expanded significantly during evolution of early land plants. Thus, some miRNA families were specific to bryophyte Physcomitrella, whereas other miRNA families were specific to higher land plants (Isam et al. 2007). It is indicated that miRNAs have evolved after the divergence between vascular plants and mosses. The evolution of miRNA genes has been accompanied with miRNA functionality change due to the process of genome-wide duplication, tandem duplication, and segmental duplication, followed by dispersal and diversification. And the process is similar to the processes that drive the evolution of protein gene families (Maher et al. 2006). It is assumed that in ancient times, miRNA played an important role in plant anti-virus defense, and novel functions came into being only after the basic requirements of survival were satisfied. However, our understanding of miRNA evolution is just at the starting point for elucidating their complex regulatory roles.

miRNAs and virus infection Viruses generate miRNAs and employ them to modulate their own gene expression as well as that of their host cells (Sullivan and Ganem 2005). At the same time, however, a viral genome can be targeted by a host miRNA, either by specific miRNAs against a particular virus or by fortuitous complementarities with the multitude of miRNAs (Simo´nMateo and Garcı´a 2006). Based on the diversity of virus families, it is reasonable to predict that there will be several categories of virally encoded miRNAs. Nonetheless, extensive cDNA cloning studies across many families of RNA viruses have failed to identify miRNAs (Pfeffer et al. 2005) which is perhaps due to the predominant role of the DNA-dependent RNA polymeraseII in biogenesis of primiRNAs (Sullivan and Ganem 2005). However, miRNAs may be produced by viral RNA-dependent RNA polymerases, especially for virus families in which genomic replication or transcription occurs in the host nucleus. The first virus exhibited to encode miRNA is EBV, a causative agent of infectious mononucleosis (Pfeffer et al. 2004)

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followed by many discoveries (Bennasser et al. 2004; Omoto et al. 2004; Cai et al. 2005; Omoto and Fujii 2005; Pfeffer et al. 2005; Samols et al. 2005; Sullivan et al. 2005). However, no conservation has been observed among the virally encoded miRNAs. Recently, an easy-to-use web interface for examining predicted viral miRNA hairpins has been established (Li et al. 2008) with an accessible viral miRNA prediction data base (Vir-Mir) at http://alk.ibms.sinica.edu.tw. Although 10 virally encoded miRNAs have been found so far, none has been identified in plant viruses. The high sequence specificity of miRNAs facilitates and promotes the selective investigations on certain viruses. Unfortunately, effective strategies for exploiting the function of virally encoded miRNAs have not been established. On the other hand, computational predictions show that these miRNAs could participate in a variety of functions: biogenesis of other small RNAs, viral DNA polymerase synthesis, viral transcription, as well as host cell apoptosis. Indeed, it has been confirmed experimentally that viral miRNAs function as suppressors through a viral mRNA encoded large T antigen (Sullivan et al. 2005). Convincing experimental evidence is available that virally encoded miRNAs were involved in counter-defense to circumvent plant defense system. This argument rests on the observation that cleavages of the early SV40 mRNAs by its own miRNAs led to the reduced expression of T antigens and sensitivity to lysis by cytotoxic T cells without reducing the yield of infectious virus (Sullivan and Ganem 2005). It is substantiated by evidence that adenovirus encodes the small VA1 RNA, analogous to a miRNA precursor. Small VA1 RNA down-regulates the host miRNA biogenesis (Lu and Cullen 2004). In this way, adenovirus circumvents the host defense system. On the other hand, viral transcripts can be regulated by host miRNAs. Naturally occurring miRNA in plants participate in viral infection. Indirect evidence for this originated from the observation that Arabidopsis mutant dcl1 showed reduced susceptibility to RCNMV infection (Dunoyer et al. 2004). The primary role of DCL1 is to process pre-miRNAs. Thus it is supposed that viruses not only suppress, but also exploit endogenous miRNA to redirect host gene expression. Interestingly, miRNAs do not detectably affect viral mRNA translation or RNA stability (Jopling et al. 2005). Implicit in this phenomenon is an assumption that the miRNAs are involved in folding of viral RNAs and/or redirecting of viral RNAs to particular sites of replication.

miRNAs and plant defense More and more evidence has shown that gene silencing is widely adopted in plant immunity. In the past, studies often

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focused on transposon or siRNA-mediated RNA silence. For instance, Tobacco mosaic virus (TMV) containing a stretch of phytoene desaturase (PDS) silenced the transcription of PDS mRNA (Carr et al. 1992). This may be the first illustration of gene silencing induced by a virus. Recently, an endogenous siRNA, nat-siRNAATGB2, has been proven to contribute to RPS2-mediated disease resistance. It repressed PPRL, a putative negative regulator of the RPS2 resistance pathway (Katiyar-Agarwal et al. 2006). Since miRNAs and siRNAs share many features in common, it is supposed that miRNAs may also be involved in silencing invaders. This was supported by the observation that siRNAs functioned as miRNAs and miRNAs interacted with mRNA in the same way as siRNAs (Doench et al. 2003). A family of Arabidopsis mRNAs encoding SCARECROW-LIKE (SCL) transcription factors is cleaved by an RNAi-like process directed by miR171 (Llave et al. 2002). In plant embryo extracts, an endogenous miRNA that lacks perfect complementarity to its RNA targets acts as a siRNA (Tang et al. 2003). In other words, the data reveals an interchangeable functional role between miRNA and siRNA. Plant virus-derived small RNAs in the gene silencing (VIGS) process were generally considered to be siRNAs. The prevalence of imperfect hairpin structure prompts a re-evaluation of their biochemical nature. In fact, many of these molecules might be akin to miRNAs, because their hairpins have greater similarity to miRNA precursors than to the perfect dsRNAs that produce siRNAs (Dunoyer and Voinnet 2005). Plant virus infections resulted in a dramatic increase in miRNA whereas virus infected vertebrate cells increased siRNA content (Bennasser et al. 2005). There have been many studies to identify plant miRNAs and numerous miRNAs have been discovered in Arabidopsis and rice (Adai et al. 2005; Bonnet et al. 2004; Floyd and Bowman 2004; Jones-Rhoades and Bartel 2004; Li et al. 2005; Li and Zhang 2005; Llave et al. 2002; Mette et al. 2002; Sunkar and Zhu 2004; Wang et al. 2004a, b; Palatnik et al. 2003; Park et al. 2002; Reinhart et al. 2002; Sunkar et al. 2005; Wang et al. 2004a, b). miRNAs have also been identified in other plant species, such as Nicotiana tabacum (Billoud et al. 2005), Zea mays (Dezulian et al. 2005), Sorghum bicolor (Bedell et al. 2005), Populus (Lu et al. 2005; Tuskan et al. 2006), Gossypium hirsutum (Qiu et al. 2007), Brassica napus(Xie et al. 2007) and Vitis vinifera (Velasco et al. 2007). Furthermore, miRNAs were predicted to play important roles in mosses Physcomitrella (Arazi et al. 2005), and unicellular green alga C. reinhardtii (Zhao et al. 2007). About 71 plant miRNA families have been identified so far. Numerous miRNAs have been predicted or validated to be involved in plant defense. For example, 9 in 48 miRNAs are related to defense in Physcomitrella. MiR1-39 targets a

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gene coding for a mucin-like protein carrying a dense sugar coating against proteolysis, which is a pivotal step in pathogen invasion. MiR160-3 acts on intracellular pathogenesis-related protein. MiR408 provides defense though interaction with the genes coding for a copper ion binding protein, and with electron transporter or Phytocyanin homolog (Isam et al. 2007). EST analysis has been used to identify plant miRNAs and 476 EST contigs were predicted to contain miRNAs. 36 EST contigs were associated with pathogen infection (Zhang et al. 2005). Approximately 70% of 130 miRNA targets were predicted to be involved in the defense response in Populus (Lu et al. 2005). In our work, we found that V. vinifera miR171e targeted blight resistance protein (RGA1) together with resistance protein candidate. Blight, caused by pathogen Botrytis cinerea, is the most devastating disease of grapevine. We also found that mir166a may target osmotin-like protein (OLP) precursor. OLP does not naturally occur in healthy leaves. In transgenic potato, two OLP genes were activated by fungal pathogen. Infection with Phytophthora infestans resulted in strong OLP expression. Over expression of a plant miRNA (miR393) resulted in the increased bacterial resistance (Navarro et al. 2006). Therefore, it is thought that plant miRNA-directed RNAi or miRNAspecified mRNA destruction determines the balance in plant defense system. All known miRNAs related to plant defense system are listed in Table 1.

Roles of miRNAs in plant-virus armament competition During evolution, the primitive plants that were subject to virus infections would have had to evolve a series of mechanisms to counteract viral infections. Of course, the defense mechanisms would have developed along an evolutionary route, following the principle that from simplicity to intricacy. One of the weapons is gene silencing. The original gene silencing discovered in plants was likely to be

Table 1 Known miRNAs related to plant defense system

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Host plants

the homology-dependent gene silencing where a perfect match is necessary. This mechanism was first discovered in plants transformed with the 54-kDa sequence of Tobacco mosaic virus (TMV) (Carr et al. 1992). It appears to be the simplest strategy to withstand invaders. However, this weapon behaved feebly because of the high mutation frequency of viruses. Perhaps plants lost the first bout. In the course of evolution, siRNA-mediated gene silencing would have likely emerged as a mechanism that protects the genetic code. An advantage of siRNA mediated silencing defense system is that the defensive signal can spread. Therefore, inoculation in one area can confer immunity on surrounding cells (Plasterk 2002). Plants thus adequately prepare their defense because a systemic response will be elicited before viruses are transmitted from the site of infection to neighboring cells and they would have had an opportunity to strengthen their defense capacity. It was confirmed by grafting experiments that silencing can be transmitted from silenced stocks to non-silenced scions (Palauqui et al. 1997). Transgenic plants, harboring untranslatable transcripts of coat protein gene from Tobacco etch virus (TEV), have been found to interfere with TEV replication (Lindbo and Dougherty 1992). In the course of this experiment, an interesting phenomenon was discovered, namely a recovery phenotype. In these transgenic plants, a systemic infection initially occurred; however, each new leaf subsequently had fewer symptoms. Eventually, virus-free leaves emerged that were completely resistant to super-infection. The possible reason of recovery phenotype was that PTGS was induced too slowly or poorly to significantly inactivate the virus, but a signal was produced and amplified in recipient cells so that the recipient cells can perform effective silencing. Another advantage of siRNAs is transitivity. It gives rise to the production of siRNAs that do not necessarily share sequence-homology with the initial target. ‘Primary’ siRNAs are perfectly complementary to targeted RNA, but ‘secondary’ siRNAs are also detectable, upstream or

miRNA

Target protein or virus

Reference

Physcomitrella

miR1-39

A mucin-like protein

Isam et al. (2007)

Physcomitrella

miR160-3

Pathogenesis-related protein

Isam et al. (2007)

Physcomitrella

miR408

Electron transporter

Isam et al. (2007)

Populus

miRNAs

Defense response

Lu et al. (2005)

Arabidopsis

miR393

Auxin signaling

Navarro et al. (2006)

V. vinifera

miR171e

RGA1

Not published

V. vinifera

mir166a

OLP precursor

Not published

Rice

Artificial miRNAs

Rice dwarf virus

Ma et al. (2004)

Arabidopsis

Artificial miRNAs

TYMV and TuMV.

Nicotiana

Artificial miRNAs

Plum pox virus

Niu et al. (2007) Simo´n-Mateo and Garcı´a (2006)

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downstream from the initial stretch (Tang et al. 2003). Transitivity ensures the immunization of naive cells before the ingress of viruses (Saumet and Lecellier 2006). It is obvious that siRNA-mediated defense systems allow the immune system to raise a massive response attack. Plants have thus made great strides in improving their antiviral systems. However, siRNA-mediated silencing is triggered only after the invader has struck. Virus infection usually starts with entry via a small wound. If the first-challenged cells are not quick enough to recognize and destroy the virus, but can send a warning message to non-infected cells, these recipient cells are stimulated to prepare their degradation mechanism. If the signal contains fragments of the virus sequence, the recipient cells are thus ready to degrade RNAs containing these sequences before the virus arrives (Waterhouse et al. 2001). However, if the virus moves ahead of the signal, it can generate an infection as soon as it enters the distant cells (Roth et al. 2004) (Fig. 1). The ultimate winner in the race is open to doubt. More than at any other time, a proactive mechanism is needed. Endogenous miRNAs exhibit this preparative feature. miRNAs that have already existed within a cell before

viruses invade help to serve as advance preparation to counteract the infection (Fig. 1). Evidence shows that miRNAs function in much the same way as siRNA duplexes in plants (Tang et al. 2003). These two separate mechanisms for target mRNA destruction work together. Endogenous mRNAs might be regulated by endonucleolytic cleavage directed by miRNA-programmed RISC complexes. On the other hand, exogenous silencing triggered by viruses, might initiate successive cycles of siRNA-mediated silence. miRNAs in plants have evolved to optimize cleavage efficiency rather than maximize complementarity to their targets (Tang et al. 2003). Three or more mismatches between a miRNA and its target RNA are permitted. It may expand the spectrum of targets. Furthermore, it may facilitate the release of the cleaved target RNAs from the RISC complex, thereby increasing the rate of enzyme turnover. To some degrees, miRNAmediated silencing might constitute reinforcement to siRNA-mediated silence. A comparison in plant anti-virus gene silencing pathways is listed in Table 2. miRNAmediated gene silencing exhibits several advantages over other gene silencing strategies: (1) proactive and long-

Fig. 1 Illustrations of siRNA-mediated and miRNA-mediated antivirus pathway. a Virus infection starts after it penetrates cell surface. The virus replicates itself in the initially infected cell and then moves into adjacent cells, spreading from cell to cell until it enters the vascular system, which allows rapid movement to distant parts of the plant. In response, the host plant initiates siRNAs silencing against the viral RNA and produces a mobile silencing signal. This signal moves along the same route that the virus takes. The plant and virus

thus enter a race. b The mobile silencing signal reaches the noninfected cells first, the virus will enter those cells only to find itself targeted by RNA silencing. The infection will then fail to become systemic. c The virus moves ahead of the signal, it can generate an infection. e The miRNAs exhibit have already been produced within cells before viruses invade. The viruses entering subsequently are targeted by miRNA-mediated silencing. 1 Dicer, 2 DCL

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1576 Table 2 Comparison of plant anti-virus gene silencing pathways

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Homology-dependent

siRNAs

miRNAs

Conservation

No

Lower

High

Matching stringency

High

Lower

Lowest

Proactive

No

No

Yes

Signal

No

Yes/exogenous

No/endogenous

acting, (2) without disruption by a non-target virus (Juan and Carmen 2006), (3) multiple targeting. On the other side, viruses struggle to counteract plant silencing defensive system. The viral silencing suppressor can help viruses at an early stage of infection. P1/HC-Pro has been found to act as a suppressor of virus-induced gene silencing through targeting RISC. A potato virus X vector containing green fluorescent protein (GFP) was constructed to induce silencing of GFP. The silencing effect was eliminated or greatly reduced when P1/HC-Pro was coexpressed with potato virus X vectors (Anandalakshmi et al. 1998). Tombus virus p19 has been identified as an efficient suppressor by sequestering viral siRNAs (Silhavy et al. 2002, 2004). In addition, the biogenesis of miRNAs was affected by p19, although no miRNA or p19 binding intermediates were found in vitro (Silhavy et al. 2002). The possible cause is the disruption of the miRNA maturation process by p19 (Papp et al. 2003). In general, viruses accomplish counter-defense by targeting RNAs (Guo and Ding 2002; Thomas et al. 2003; Qu et al. 2003) or protein (Anandalakshmi et al. 2000). Therefore, expression of host genes is modified or silenced (Dong et al. 2003). Recent findings suggest that silencing suppressors can contribute to viral symptoms in two ways: helping virus accumulation indirectly and modifying endogenous short-RNA-regulated pathways directly (Silhavy and Burgya´n 2004). Plant defense systems are elegant examples of how nature can find highly efficient solutions to the problems it faces (Waterhouse et al. 2001). Overall, it can be described as a co-evolution of defense and counter-defense mechanisms between the host plant and the invading virus. It is likely that small RNAs have existed since the very beginning and cooperated with each other to optimize the effect. miRNAs, as endogenous small RNAs, play versatile roles in a plant’s defense system, but their functions in anti-virus defense are far from being fully understood.

The application of miRNAs in plants’ anti-virus defense Plants do not possess an antibody-based immune system analogous to that in animals (Waterhouse et al. 2001). However, the cross protection discovered in 1920 gave scientists an impetus to seek the reason why plants can be protected from severe virus by prior infection with a mild

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strain of a closely related virus. Considerable work has been focusing on this mechanism that is just beginning to be understood. Antisense suppression, VIGS, TGS and RNAi were thought to be involved in the process and they have been brought into play in plant anti-virus biotechnology individually or cooperatively. In addition, miRNAmediated pathway was newly discovered strategy to suppress plant viruses. Many of the details and ramifications have yet to be determined, but the potential application of miRNAs to contend with the virus is obvious. The potential value of miRNAs as antiviral agents in plant biotechnology was manifested in the following experiment. A hairpin RNA (hpRNA), the precursor of miRNA, was constructed from a segment of Rice dwarf virus (RDV). Then the hpRNA was inserted into a vector. The transgenic plants expressing the vector displayed high resistance or attenuated viral symptoms (Ma et al. 2004). Endogenous miRNAs have been shown to target engineered plant viruses harboring the target sequences of miRNA. A member of potyvirus family, Plum pox virus (PPV), was constructed to bear Arabidopsis miRNA (miR171, miR167, and miR159) target sequences. As a result, the engineered virus was affected by Nicotiana clevelandii and Nicotiana benthamiana miRNA (Simo´n-Mateo and Garcı´a 2006). Furthermore, multiple-targets miRNAs can impact on several viruses. miRNA precursors were designed to contain complementary sequences with two viruses, Turnip yellow mosaic virus (TYMV) and Turnip mosaic virus (TuMV). The transformed Arabidopsis with the recombinant miRNA precursors exhibited specific immunity to these viruses (Niu et al. 2007). Moreover, miRNA-mediated defense has been demonstrated for two very different plus-strand RNA viruses (Garcı´a and Simo´n-Mateo 2006). It suggested that this approach should be of broad utility. It is assumed that the effects of the miRAN targets cloned to viruses depend not only on their nature, but also on their inserted positions (Simo´n-Mateo and Garcı´a 2006). It was significant that some hits have a propensity to be more effective than others. An analysis of flanking sequences reveals that miRNA silencing machanism or processing is somehow influenced by the flanking sequence rather than by the miRNA sequence alone. The possible reason is that RNA folding impacts on the binding sites between miRNAs and the targets. Therefore, the insertion sites and the flanking sequence must be scrutinized when

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miRNA-directed silencing is devised. It is certain, however, that miRNA-directed anti-virus biotechnology will make great strides if the precise mechanism can be defined. There are several advantages of using miRNAs over homology-dependent gene silencing and siRNAs: (1) Fewer off-target effects. Given a short sequence of miRNAs, the selection of antiviral amiRNAs that have no complementary host target sequences is feasible when a complete genome sequence is available. (2) Highly RNA promoter-compatible. (3) Environmental biosafety. No non-target viruses complement or recombine with transgenic plants with viral sequences. (4) Stable in vivo, usage of miRNAs is adapted at low temperature. The following areas have the potential for application of miRNAs in plant anti-virus defense: (1) Analyzing the function of viral suppressor in the process of gene silencing. (2) Designing and developing novel miRNA-mediated gene therapy. (3) Modifying plant physiological properties to enhance their anti-virus capacity. (4) Developing lossof-function transgenic plants. Still, current research faces several problems. Firstly, broad-spectrum miRNAs are difficult to construct due to high sequence divergence of plant viruses. Moreover, the permanence of the miRNA effect is a challenge owing to the resistant mutants. Furthermore, the application of miRNAs against plant viruses suffers from immunity feebleness. Transgenic plants expressing single miRNA may meet with strong virus pressure when they are grown under field conditions. In addition, transgenic plants are significantly more variable in field than in greenhouse conditions.

Concluding remarks As our understanding of plant miRNA genome and function grows, the application of miRNAs to counteract plant viruses will be at the cutting edge. The overall picture demonstrates that miRNA was adopted as an ancient tool in plant defense system. The roles of miRNA in the competition between plants and viruses should be well illustrated. The challenge is to clarify the full extent of miRNA functional diversity in plant-virus interaction. More informative approaches are needed in order to solve the mystery of miRNAs involved networks. Although this field is still in its infancy, the idea that miRNAs can be used in the therapy of plant viral infection is certain. If smart miRNAs can be used appropriately, a new avenue of biotechnology aimed at achieving enhanced plant defense will be opened. It will be yet another example of ingenious use of simple tools to solve complex problems in nature. In addition, the concepts discussed here might not be restricted to viruses. They could, in principle, apply to other types of pathogens that employ miRNAs as a part of their infection strategy.

1577 Acknowledgments We would like to thank all colleagues who have done work on phytopathology, miRNAs and related fields. We are grateful to Prof. Prakash Kumar for valuable advice on revision of the manuscript. We acknowledge the colleagues whose work in this rapidly changing field was not directly cited in this review due to space limitations and timing.

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