RNA mediated silencing technology has now become the tool of choice for induction of virus resistance in plants. A significant feature of this technology is the presence of double-stranded RNA (dsRNA), which is not only the product of RNA silencing but also the potent triggers of RNA interference (RNAi). Upon RNAi induction, these dsRNAs are diced into short RNA fragments termed as small interfering RNAs (siRNAs), which are hallmarks of RNAi. Considerable resistance in transgenic plants against viruses can be created by exploiting the phenomenon of RNAi. In the current chapter, generation of potato virus Y (PVY) resistant potato and sugarcane mosaic virus (SCMV) resistant sugarcane by CEMB has been quoted as an example.
We are in the dawn of a new age in functional genomics driven by RNAi methods. RNA interference (RNAi) refers to a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. It is one of the most exciting discoveries of the past decade in functional genomics and is rapidly becoming an important method for analyzing gene functions in eukaryotes and holds promise for the development of therapeutic gene silencing and which is therefore currently the most widely used gene-silencing technique in functional genomics.
2. Need for resistance
Agriculture sector of any country strengthen the economy by contributing in its gross domestic product (GDP). In Pakistan, the major agricultural crops include cotton, wheat, rice, sugarcane, potato and tomato etc. All of the above mentioned crops has great potential for yield and contribute 24 % in gross domestic product (GDP) of Pakistan economy . There is a major gap in the actual yield potential of each crop with respect to its harvested yield, possible reasons include disease attack, environmental damages and in some cases lack of quality seed. Disease attack as being the most common cause include infections by pathogens like viruses, bacteria and insects etc. Viruses can cause most devastating effects due to their systemic infections and hence decrease crop productivity in primary infection and reduce seed quality for subsequent use through persistent infection. Viral epidemics are often associated with the emergence of a new form of the viral strain or a new form of vector.
Viruses are a major threat to agriculture all over the world. Up till now, more than 1200 plant viruses have been reported which include 250 of those viruses that cause signiﬁcant losses in crop yield . In nature, viral particles exist as obligate parasites which consist of hereditary material packed in a thick layered coat and completely depend on host cell throughout their life cycle. Viruses utilizes host resources like nucleic acid, amino acids and certain proteins for their replication and survival, thus disturbing host plant metabolism to a considerable extent. Most of the infecting plant viruses are ssRNA viruses like sugarcane mosaic virus, potato virus Y etc. In an infected plant, virus accumulation goes higher with increased progeny rate through its replication. The spread of the virus in a plant is achieved through its movement from infected cell to healthy one via plasmodesmata while long distance movement occurs through phloem. Entry of virus in plant usually occurs through physical injury like wound etc or via certain viral vectors like aphid, fly etc.
Cotton (Gossypium hirsutum) as being commonly known as ‘white gold’ is an important cash crop in many developing countries including Pakistan. It is a natural fibre and has many uses in industries. It accounts for 8.2 percent of the value added in agriculture and about 2 percent to GDP of Pakistan. The yield of the crop is severely affected by the viruses including geminiviruses (leaf crumple and leaf curl) and tobacco streak virus etc. These viruses can cause severe losses when infections occur on young plants; some infect cotton yield while others affect lint quality as well .
Potato (Solanum tuberosum) is the world's major food crop and is one of the leading vegetables. Viruses are a serious problem, not only because of effects caused by primary infection, but also because the crop is vegetatively propagated and the viruses are transmitted through the tubers to subsequent generations. Potato virus Y (PVY) is probably the most damaging and widespread virus of potato and is found wherever potato crops are grown, where losses are reported upto 10 to 90% . PVY is transmitted through aphids.
Sugarcane (Saccharum spp. hybrid) is among the top 10 food crops of the world, and yearly provides 60% to 70% of the sugar produced around the world . Yield harvested by the farmers of Pakistan is very low whose main cause is mosaic disease of sugarcane which continues to be a potential threat to the sugarcane production. It is a very common disease in all the major sugarcane growing regions, because of the perpetuation of the disease virus through vegetative propagules. Sugarcane mosaic virus (SCMV) is reported to infect sugarcane naturally and can cause severe losses to the farmers and lesser production to the industry [6,7]. Aphids are the vector for transmission of the disease. Seed produced by infected cane can also transmit the disease.
Tomato (Lycopersicum esculentum) ranks among the most widely grown vegetables all over the world. In general, viral diseases are not a routine problem in most tomato plantings but incidence of some viruses including tomato spotted wilt virus, tomato leaf rolling, tobacco mosaic virus and single and double virus streak viruses has devastating impact on crop yield with losses of upto 100% have also been reported . Tobacco mosaic virus is one of the most stable viruses known because it is able to survive in dried plant debris as long as 100 years.
Various control measures have been taken to overcome losses caused by plant viruses which are expensive and also inadequate. Biotechnologists have developed and adopted several strategies for virus resistance in crop plants. These include cross protection, pathogen derived resistance and more recently RNA interference. Therefore, the development of virus resistant varieties seems the only economically feasible way to control viruses . Today, use of resistant varieties has been advocated as the most promising and least expensive method of viral disease suppression. With the appealing results of RNAi in silencing target genes of attacking virus, RNAi seems to have potential for creating virus resistant crops.
A plant is said to be resistant if it has the ability to suppress viral disease symptoms by inhibiting its replication or by blocking the virus expression. Resistance mechanism in plants may be either protein mediated or RNA mediated, however final outcome of both is reduced accumulation of virus in the host plant. Acquired resistance could be either high almost reaching immunity with no disease symptoms or moderate to low where mild symptoms of particular viral disease can be seen. In contrast, when a viral infected plant shows normal growth rate with a good yield along with milder symptoms of disease, it is said to be a tolerant plants. In this case, the host plant supports multiplication of virus rather than blocking its replication [10,11].
3. History of virus resistance in transgenic plants
When a plant encounters virus, it reacts naturally through hypersensitive response (HR) and extreme resistance response (ER) which induces the production of secondary metabolites termed as response elements in plants. These response elements include elevated levels of ethylene, jasmonic acid, salicylic acid, nitric oxide and increased rate of ion flux, in combination these factors block the virus entry and /or helps eliminate the virus (figure 1).
The acquired virus resistance mechanisms in plants are of two types: a) gene silencing independent virus resistance and b) gene silencing dependable virus resistance via Post Transcriptional Gene Silencing (PTGS). The first includes coat protein-mediated, movement protein-mediated and replicase protein-mediated resistance, while second includes pathogen-derived resistance, antisense RNA mediated resistance and RNA-mediated resistance. PTGS is an evolutionary conserved mechanism in plants against potential harms by viruses and transposons. In this process, a plant defends itself by exploiting the requirement of plant RNA viruses to replicate using a double-stranded, replicative intermediate (dsRNA). The double-stranded RNA produced is cleaved into approximately 21 nucleotide fragments by the Dicer enzyme . Evidence suggest that transgene loci and RNA viruses can generate double-stranded RNAs which are similar in sequence to the transcribed region of target genes, which further undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs.
The first approach made by plant agronomists was the inoculation of susceptible plant with a milder strain of the target virus. This technique was named as cross protection and was employed on crops like tomato, papaya and citrus [13-15]. Scientists were met with success as considerable resistance was achieved in transgenic plants through employment of this approach but the success was accompanied with a major drawback that the milder strain of the virus providing protection to one crop may cause serious diseases on varieties growing nearby.
To compensate the drawback of cross protection, pathogen derived resistance (PDR) based strategies were employed. These are based on the insertion of resistant genes that are derived from the pathogen (virus) into the host plant. Resistance was achieved by expressing viral genes in plants including coat protein, movement protein and replicase protein gene, each of them targets at a step crucial to virus replication. Coat protein gene is responsible for viral uncoating and is involved in virus replication , movement protein is crucial for cell to cell movement of the infecting virus  whereas the Rep protein is involved in virus replication and its genome integrity [18,19]. Resistance was either due to protein accumulation (coat protein mediated resistance, movement protein mediated resistance and replicase protein mediated resistance) or because of accumulation of small RNA sequences (replicase mediated resistance).
Uptill now, scientists have made considerable successful attempts to generate virus resistant transgenic plants by employing PDR concept [20, 21]. For example, virus-resistant potato varieties having PVY coat protein (CP) or P1 gene sequences has been reported in numerous studies [22-27]. Biotechnologists employed various genes of PVY and have met with mixed success in engineering PVY resistant transgenic potato plants [22,28-30,24,31,25,32]. In another study, the presence of the movement protein (pr17 protein) was reported to create resistance in transgenic plants against luteovirus Potato leaf roll virus .
Although pathogen derived resistance strategies hold promise for upto 90% resistance against target virus and are being employed still to date but some remarkable and potential threats are also associated with the use of this technology. The major one includes; the expression of a gene fragment derived from virus in transgenic plant confers resistance to particular virus but at the same time also raises environmental safety concerns regarding the constitutive expression of viral genes. It is supposed that infecting virus can interact with expression product in transgenic plants and can potentially modify the biological properties of the existing virus, ultimately leading to creation of new virus species which have novel pathogenic properties, host range and altered transmission specificity. In the initial experiments, the virus resistance was based on protein expression but resistance was neither so stable nor effective as compared to the resistance achieved through RNAi.
Among pathogen derived resistance strategies, antisense RNA complementary to part of the viral genome proves to have potential utility for protecting plants from systemic virus infection . Antisense RNAs refer to small untranslatable RNA molecules that pair with a target RNA sequence on homology basis and thereby exert a negative control on interaction of target RNA with other nucleic acids or protein factors. Further, RNase H cause an increase in rate of degradation of double stranded RNA . Antisense RNA technology was quickly adopted by plant researchers because other approaches like homologous recombination and gene-tagging mutagenesis used were based on reverse genetics and also these were not applicable in plants nor these were well developed. This background makes antisense RNA-mediated suppression more powerful tool for transgenic research and also for the development of commercial products .
4. RNA silencing
The development of the concept of pathogen derived resistance gave rise to strategies ranging from coat protein based interference of virus propagation to RNA mediated virus gene silencing. Virus resistance is achieved usually through the antiviral pathways of RNA silencing, a natural defense mechanism of plants against viruses. The experimental approach consists of isolating a segment of the viral genome itself and transferring it into the genome of a susceptible plant. Integrating a viral gene fragment into a host genome does not cause disease (the entire viral genome is needed to cause disease). Instead, the plant’s natural antiviral mechanism that acts against a virus by degrading its genetic material in a nucleotide sequence specific manner via a cascade of events involving numerous proteins, including ribonucleases (enzymes that cleave RNA) is activated. This targeted degradation of the genome of an invader virus protects plants from virus infection.
Transcriptional Gene Silencing (TGS)
In plants, silencing is of two types: transcriptional and post transcriptional gene silencing. In both types, the inactivated genes are in trans position as homologous genes upon interaction reside on opposite chromosomes. TGS and PTGS differ from each other with respect to the underlying mechanism they exhibit. TGS requires sequence homology between promoters as compared to PTGS which require homology between coding region of the interacting genes. In TGS, an inactive allele residing on one chromosome can render another allele silenced. The mechanism behind transcriptional gene silencing is suggested to be DNA-DNA interaction which is thought to play an important role [37,38]. In other studies, it was proposed that RNA molecules interact with DNA and subsequently induce DNA methylation which then leads to gene silening [39-42], however it is not clear whether methylation of DNA alone is sufficient for silencing or not. It is proposed that DNA methylation in promoter region has a strong negative effect on interaction of certain transcription factors with promoter. Possible mechanism of TGS is depicted in figure 3.
Post Transcriptional Gene Silencing (PTGS)
‘RNA interference’ is a conserved mechanism of post transcriptional gene silencing (PTGS). It has rapidly gained favor as a “reverse genetics” tool to knock down the expression of targeted genes in plants. The term RNAi was coined in 1998 by Fire and Mello to describe a gene-silencing phenomenon based on double-stranded RNA . PTGS mechanism controls processes including development, the maintenance of genome stability and defense against molecular parasites (transposons and viruses). Several reports pointed out that PTGS in plants is strictly linked to RNA virus resistance mechanism [44-46].
5. Mechanism of RNAi/PTGS
RNAi (RNA interference) is a natural defense pathway evolved in plants against viruses and potential transposons. It is a cellular pathway in which target sequences are degraded on homology basis at mRNA level by small RNAs, thereby preventing the translation of target RNAs. In plants, two functionally different RNAs; microRNA (miRNA) and small interfering RNA (siRNA), have been characterized. The miRNAs are small 21-26nt long dsRNAs that are genome coded and are endogenous to every cell. Structurally, they comprised of a stem region which is double stranded and a loop region which is single stranded. The miRNAs generated from endogenous hpRNA precursors and are basically involved in the regulation of development . On the other hand, siRNAs are generated from long dsRNA and are involved in defense through RNA interference [48, 49].
RNAi is an immune system in plants which is directed against viruses . Upon viral attack, long dsRNAs are produced from the replication intermediates of viral RNAs that act as substrate for an endonuclease termed Dicer which is located in the cytosol . Dicer recognizes these dsRNAs and cleave them into duplex siRNA (21-25 nt) . The siRNA duplex comprised of two strands; strand complementary to target mRNA is guide strand and other is passenger strand. The guide strand of short siRNA duplex is incorporated into the RNA-induced silencing complex (RISC) and then siRNA programmed RISC degrade viral RNA. As the RISC complex encounters a foreign mRNA which could be of virus origin, it has two consequences. 1) If the homology of guide strand and target mRNA is 100%, then perfect complement form between them resulting in mRNA cleavage and subsequent degradation or 2) in case of imperfect complement, where few mismatches exist between guide strand of RISC and target mRNA, translation of target mRNA is inhibited (figure 4).
Same mechanism operates in microRNA triggered gene silencing. miRNAs processed from stem loop precursors (shRNA and/or hpRNA) and requires Dicer activity  followed by RISC assembly and subsequent degradation of homologous RNA in a sequence specific manner.
RISC is a combination of Dicer (an endonuclease enzyme), some accessory proteins namely argonaute (ago1, 4, 6, 9; catalytic endonucleases) and RNA binding proteins (RBP), and some trans-acting RNA-binding proteins (TRBP) [54,55].
Stability of RNAi induced silencing is based on enzymatic methylation of siRNA. This reaction is catalyzed by the enzyme methyltransferase (HEN1) which methylates the siRNA at 3’ end, hereby preventing it from oligouridylation and subsequent degradation .
6. Systemic spread of RNAi
When RNAi is induced at one site in an organism including plant, a mobile signal is generated which spread cell to cell and systemically throughout the organism [57-59,43,60,61] and make RNAi response obvious in distant tissues of the plant. This silencing signal moves inside plant either through the intercellular spaces called plasmodesmata or through the phloem as shown in figure 5 [59,60]. Presence of a mobile signal has been proposed to be an integral part in systemic spread of silencing. The first evidence of the presence of a mobile silencing signal came from the study of Agro infiltration assay or particle bombardment in development of transgenic tobacco plants [59,60,62]. Subsequently, in silenced tissues of Agro-infiltrated plants, T-DNA or Agrobacterium was detected which suggests that mobile signal is responsible for propagation of silencing from one tissue to another  and this signal can also cross graft junction [59,60,62]. Candidates proposed to be responsible for mobile silencing signal involve siRNAs, Aberrant RNAs and dsRNA .
Conclusively, transgenic approach mediated by RNAi pre-programmed an existing antiviral defense in plants [21,64-66]. Plant viruses are the strong inducers of RNAi as well as a target. The simplicity and specificity of RNAi has made RNAi a routine tool for the generation of virus resistant crops.
7. Effective RNAi inducers
In general, gene silencing has proven fruitful with both sense- and antisense transgenes in plant cells [67,68]. An RNA molecule that contains a fragment of a sense strand, an antisense strand and a short loop sequence between the fragment making a tight hairpin turn is termed as short hairpin RNA (shRNA) which has the ability to suppress the expression of desired genes via RNA interference . Silencing can be more efficiently achieved by utilizing shRNA cassettes [70-72] which usually include a specific plant promoter and terminator sequences to control the expression of inversely repeated sequences of the dsRNA. Upon subsequent delivery of shRNA cassette in the plant cells, dsRNA molecules comprised of a loop (single-stranded) and a stem region (double-stranded) are formed. Further, stem region is used by Dicer as a substrate and trigger RNAi mechanism [72-74]. RNA silencing mediated by the use of shRNA cassette enforces stable and heritable gene silencing  as it utilizes the specific promoter to ensure that the shRNA is always expressed. Another reason which justify that the silencing efficiency can be more powerful when using shRNA cassette is due to the fact that dsRNA are being fed into a later step in the silencing pathway where they act as a substrate for Dicer (RNaseIII like enzyme) and therefore bypasses the step in which dsRNAs need plant encoded RdRps for their production .
Practically in development of virus resistant transgenic plants, specific hairpinRNA expression constructs have been designed for transformation. In this strategy, small dsRNAs which are hallmark of PTGS, are produced from the transformed construct and ultimately induce silencing. Scientists have used hpRNA construct for silencing of viral gene in potato and obtained efficient silencing results accompanied with production of siRNA . Similarly, some others have compared various constructs in terms of their silencing potential and confirmed that most efficient and strong silencing in tobacco can be achieved through the expression of an intron containing construct, which trigger PTGS . However, in another study where PVY resistant potato plants were obtained through CP gene expression, evidence for existence of both protein and RNA mediated mechanisms was verified .
While considering the appealing outcome of RNAi in development of virus resistant transgenic plants as reviewed in this article and the use of hairpin RNA for strong silencing, production of transgenic potato resistant against potato virus Y and sugarcane plants resistant against sugarcane mosaic virus developed by  at Centre of Excellence in Molecular Biology (CEMB), University of the Punjab has been quoted as an example in following chapter.
8. Development of PVY and SCMV resistant transgenic plants
Tabassum et al.  have developed PVY and SCMV resistant potato and sugarcane plants respectively through siRNA technology by targeting capsid protein gene of respective virus. In the study, the respective plant was equipped with shRNA cassette that reacts continuously against invading virus specifically, thus resulting in degradation of viral mRNA in a sequence-specific manner. Specialty of this shRNA cassette is that it contained screened siRNA (the one most efficient in in-vitro experiments) out of bulk. The 22nt long siRNA was used as core sequence in shRNA cassette while loop sequence and flanking sequences were taken from highly active regulatory microRNA of respective host plant.
Initially, for screening of siRNA out of bulk, a strategy based on transient transfection assay was optimized in mammalian cell line (CHO). mRNA knockdown efficiency of capsid protein gene of target virus was analyzed by real-time PCR. In case of PVY capsid gene, one specific siRNA out of a total six was found to be the most effective for knockdown of respective mRNA in transfected CHO cells by up to 80-90%. Data obtained showed that all six siRNAs used reduced the mRNA expression of target gene to some extent but only siRNA1 significantly reduced CP-PVY mRNA expression by up to 12.25 fold and, as is clearly shown in figure 6, expression was almost diminished or very faint in cells transfected with siRNA1 as compared to the control where scrambled siRNA was transfected. The remaining siRNA knockdown values were: siRNA 2 - 7x decrease ; siRNA 3 - 8x decrease; siRNA 4 - 10.8 x decrease; siRNA 5 - 9x decrease and siRNA 6 - 10x decrease. These values were based on Ct values obtained from real-time PCR studies .
Similar findings were met when knockdown in mRNA expression of CP-SCMV was studied in-vitro through transient transfection assays. As clear from figure 7, siRNA1 reduced the mRNA expression of target gene by upto 96%, while inhibition by siRNA2 was 46%, siRNA3 and siRNA4 inhibited target gene mRNA expression upto 50% and 77% respectively (figure 7).
Subsequently, the screened siRNA for both viruses was used in shRNA cassette which is thought to synthesize target specific siRNAs that continuously guard the plant against respective viral attack. shRNA cassette cloned in pCAMBIA1301 vector and transformed in potato and sugarcane through Agrobacterium- and particle bombardment method respectively. Results were compared with control non-transgenic plants. Figure 8 and 9 depicts the results, clearly indicating that in transgenic potato having shRNA1 cassette integrated in them, mRNA knockdown was upto 96% whereas in transgenic potato plants having shRNA4 cassette in them, PVY knockdown was upto 57% as compared to control where PVY infection was maximum.
Similarly, in transgenic sugarcane plants, shRNA1 reduced the mRNA expression of SCMV to lesser extent with 30% reduction only while shRNA4 caused maximum knockdown of 95% as compared to the control non-transgenic sugarcane plant.
In conclusion, we have developed transgenic potato and sugarcane plants that were highly resistant against PVY and SCMV infection respectively. This resistance was because of the shRNA cassette integrated in them that is targeted against capsid protein gene of each virus. These shRNAs are supposed to create long-term targeted gene inhibition in cells and whole plant. Our shRNA construct designing was based on the hypothesis that if we express potentially effective screened siRNA in hairpin form which is further combined with the power of most active regulatory microRNA in respective plant, the level of resistance will be far more effective. Applying this theme, we were able to obtain transgenic potato and sugarcane plants where resistance level against targeted virus was upto immunity.
One important aspect of this strategy in engineering PVY-resistant plants is the fact that the integrated shRNA sequence is not of viral origin nor it is translated into a protein. Moreover, the actual RNA transcript is almost undetectable because it gets cleaved quickly in small fragments through RNAi pathway. These two features limit the environmental risks of this strategy, such as trans-encapsidation or recombination of the transgene with an incoming virus.
RNAi (RNA interference); PVY (Potato virus Y); SCMV (Sugarcane mosaic virus); PTGS (Post Transcriptional Gene Silencing); siRNA (small interfering RNA); shRNA (short hairpin RNA); ssRNA (single stranded RNA); dsRNA (double stranded RNA); RdRp (RNA dependent RNA polymerase); hpRNA (hairpin RNA).