In December 2019 a novel coronavirus was detected in Wuhan City of Hubei Province-China. Owing to a high rate of transmission from human to human, the new virus called SARS-CoV-2 differed from others by its unexpectedly rapid spread. The World Health Organization (WHO) described the most recent coronavirus epidemic as a global pandemic in March 2020. The virus spread triggered a health crisis (the COVID-19 disease) within three months, with socioeconomic implications. No approved targeted-therapies are available for COVID-19, yet. However, it is foreseen that antibody-based treatments may provide an immediate cure for patients. Current neutralizing antibody development studies primarily target the S protein among the structural elements of SARS-CoV-2, which mediates the cell entry of the virus through the angiotensin converting enzyme 2 (ACE2) receptor of host cells. This review aims to provide some of the neutralizing antibody development strategies for SARS-CoV-2 and in vitro and in vivo neutralization assays. In December 2019, in Wuhan City of Hubei Province-China, a novel coronavirus (coronaviridae family) was detected. Coronaviruses are members of a wide group of viruses causing various diseases ranging from flu to more extreme diseases like severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The new virus called SARS-CoV-2 differed from others by its unexpectedly rapid spread due to a high rate of transmission from human to human. There are currently no approved targeted therapies available for COVID-19. Researchers worldwide are exploring COVID-19 prevention strategies and therapeutic options, including convalescent plasma, monoclonal antibodies, vaccines, peptides, interferon, small molecule drugs, as well as exploring the repurposing of proven drugs . Vaccination may provide a strong and sustainable protection, however, vaccine development is a long and challenging process, and vaccination is only useful in a preventive environment. On the other hand, an antibody-based therapy can provide immediate effect for patients.Neutralizing antibodies (NAbs) target viral surface proteins for blocking the attachment of virus to host cell . Therefore, in SARS-CoV-2 studies, amongst all structural proteins, neutralizing antibodies primarily target the S (spike) protein, which mediates entry into cells. The structural protein S is a transmembrane glycoprotein that has 2 functional subunits: the subunit S1 that is involved in cell attachment and the subunit S2 that mediates cell membrane fusion . S1 also breaks down into 2 domains, a receptor-binding domain (RBD) and an N-terminal domain (NTD). The S protein binds the human angiotensin converting enzyme 2 (ACE2) receptor through its S1 subunit. SARS-CoV-2 appears to be using the same receptor, ACE2, for cell entry as SARS-CoV with a 10 to 20-fold higher affinity . As shown in Table, all currently developed anti-SARS-CoVNAbs target the S protein, predominantly target the RBD, while some target regions in the S2 subunit or the S1/S2 proteolytic cleavage site. S1 RBD is the most crucial target for SARS-CoV NAbs, which may interrupt the interaction of RBD and its ACE2 receptor .Here in this review, we discuss reverse engineering from convalescent plasma, classical hybridoma technology, human hybridoma technology, phage display technology, and mammalian cell surface display technology to develop human antibodies, humanized antibodies and human scFv and single domain antibodies against SARS-COV-2 . Later we provide brief summary of in vitro and in vivo neutralizing assays including animal models for SARS-CoV-2. Although there are many projects carried out in many laboratories around the World, currently, there are no approved drugs, vaccines, NAbs, or antiviral agents targeting coronavirus, and they may not be available in a short time . On the other hand, the rising number of patients recovering from COVID-19 with a high neutralizing antibody titer day by day highlights convalescent plasma therapy as a promising alternative for COVID-19 treatment .For more than a century, convalescent plasma (CP) therapy, which is traditional passive immunotherapy, has been used to prevent and treat the outbreaks of many infectious diseases which have been reviewed previously . Some clinical CP therapy experiments have already been reported on SARS-CoV-2. In China, 245 COVID-19 patients received pilot convalescent plasma therapy in February, and 91 cases demonstrated improvements in terms of clinical indicators and symptoms. Duan et al. (2020) reported the results of CP transfusion on 10 severe adult cases confirmed by a real-time viral RNA test. A single dose of 200 mL CP derived from 39 recovered donors having neutralizing antibody titers above 1:640 was transfused to the patients, and their clinical symptoms such as the oxyhemoglobin saturation, the level of neutralizing antibodies, the lymphocyte counts and the decrease of C-reactive were significantly improved. Shen et al. (2020) reported another CP transfusion study on 5 severe COVID-19 patients who were receiving mechanical ventilation during treatment, and all of whom had received antiviral agents and methylprednisolone. Following the transfusion of CP with a neutralizing titer more than 1:40, the body temperatures decreased within 3 days. Within 12 days, the SOFA scores were reduced, the PAO2/FIO2 ratios were increased, and the viral loads became negative. Neutralizing antibody titers of all 5 patients increased by scores ranging from 80 to 320. For CP therapy evaluation, the level (titers) of neutralizing antibodies against SARS-CoV-2 virus is critical, and the plasmas containing NAbs must be obtained from recovered patients when the titers of NAbs reach their peak after the onset of disease. Wu et al. (2020) screened plasma samples collected from 175 COVID-19 recovered patients using pseudotyped lentiviral vector based neutralization assay. The domain RBD, the subunit S1, and the subunit S2 of SARS-CoV-2 were used in ELISA to determine the level and time-course of spike-binding antibodies in plasma. They revealed that SARS-CoV-2 specific antibodies were detected from the day-10-15 after the onset of disease and remained thereabouts afterward. The titers of patients varied depending on age. Elderly and middle-aged patients had significantly higher plasma-NAb rates (P < 0.0001) and spike-binding antibodies (P = 0.0003) than young patients.There are also some known and theoretical risks of passive administration of convalescent sera. Transfusion transmitted infections (TTI), allergic transfusion reactions like serum sickness; transfusion associated circulatory overload (TACO), transfusion related acute lung injury (TRALI) are the known risks of convalescent sera transmission. While, TRALI is of particular concern in severe COVID-19 given potential priming of the pulmonary endothelium the risk of TRALI is less than 1/5000 transfused units. With the development of modern blood banking methods, the risk of accidental transmission of known infectious agents or triggering transfusion reactions becomes very low . There is a theoretical risk of antibody-dependent enhancement phenomenon of infection after transfusion. ADE refers to a process in which antibodies that target one serotype of coronavirus could enhance infection to another viral serotype. Wan et al. (2020), showed a novel mechanism for ADE in which a NAb that binds coronavirus surface spike protein like a viral receptor, activates spike conformation and mediates viral entry into receptor-expressing IgG Fc cells via canonical viral receptor-dependent pathways. Also, it is still unknown to what extent convalescent plasma represses the development of a natural immune response, especially when administered for prophylaxis and makes such individuals vulnerable to reinfections. Risk-benefit assessment must be investigated carefully and must be supported with scientific data. Although CP therapy is a fast and effective form of treatment that can be applied in difficult situations, is not a real solution for diseases. Since it is associated with the amount of antibody titers in the blood of patients who recover, which is not an infinite source. Therefore, reverse engineering is needed for the development of neutralizing antibodies. Ju et al. (2020), are the first group which characterized 206 RBD-specific human mAbs isolated from single B cells by FACS from eight SARS-CoV-2-infected patients. They have cloned the genes of these B cell into an expression vector for further analysis. Then they showed the potential binding and neutralizing effect of the antibodies by surface plasmon resonance assay and neutralization assay with pseudovirus and the SARS-CoV-2 virus. They found 2 potent SARS-CoV-2 neutralizing human monoclonal antibodies (P2C-1F11 and P2B-2F6). An ideal alternative to hyperimmune sera is monoclonal antibodies owing to their specific pharmacological and safety profiles . Hybridoma technology allows the production of monoclonal antibodies against specific target antigens, in large quantities in the laboratory environment. Classical hybridoma technology includes the vaccination of mice with specific antigens and the development of hybridoma cells by the fusion of antibody-producing B lymphocytes and immortal myeloma cells. The fusion merges the cytoplasms of the 2 beneficial somatic cells with the help of a chemical agent (polyethylene glycol) . Effective immunization is essential to obtain a strong and target-specific antibody response from mice. Immunization is done either by using the whole/a part of an antigen or antigenic peptides for which immunogenicity is enhanced by conjugating them to a larger and immunogenic carrier molecules such as KLH, BSA or ovalbumin . B cells that produce antibodies in the spleen and lymph nodes of animals, immunized with the target antigen, are fused with myeloma cells to form immortal antibody-producing cell lines. The basis of monoclonal antibody technology is to ensure the recognition of a single epitope and the production of antibodies by hybrid cell lines. Therefore, the antibody activities after fusion are detected by indirect ELISA. Selected hybridomas are subjected to 3 rounds of cloning using limited dilution method. Cross-reactions are investigated by testing the antibody activity against similar viruses, enzymes and proteins, especially blood serum proteins, to determine the specificity of the antibodiesIn recent studies, chimeric antibodies, containing both human and mouse antibody sequences, have been developed by hybridoma technology to treat and prevent the new types of human coronavirus MERS-CoV and SARS-CoV-2 associated diseases . Wang et al., 2020, screened the supernatants of 51 SARS-S hybridomas derived from immunized transgenic H2L2 mice that encode chimeric immunoglobulins (human variable regions and rat constant regions) against SARS-CoV-2 S2-S1 region. Four of the supernatants displayed cross-reactivity with the subunits S2 and S1 of SARS-CoV-2. One of these hybridoma supernatants (47D11) revealed neutralizing activity in pseudotyped virus neutralization assay. The chimeric 47D11 was then reformatted into a fully human antibody. They hypothesized that this antibody is neutralizing both SARS-CoV and SARS-CoV-2 through a different binding region than the expected RBD-ACE2 binding. Xiong et al. (2020) reported that SARS-CoV-2 spike RBD domain specific antibody was generated from immunized BALB/c mouse with SARS-CoV-2 spike RBD domain protein. With this study, mAbs against SARS-COV-2 RBD were produced using hybridoma technology. Using neutralizing mouse antibodies developed by the hybridoma technology may cause unfortunately the production of antimouse antibodies in humans (HAMA). Therefore, an antibody humanization step is necessary to reduce HAMA responses . However, with the increased number of convalescent patients, the development of human mAbs from the covalescent patients has gain interest because no HAMA response is expected and the antibody humanization step is not needed.There are many methods available for the development of human monoclonal antibodies (mAbs), including the transformation of Epstein-Barr virus (EBV), the use of phage-display libraries, the mammalian cell systems (i.e. CHO or NS0 transfectomas) to recombinantly produce mAbs by the immunization of transgenic mice carrying human Ig genes (xenomice), the molecular techniques focused on antigen-specific B cell isolation of Ig genes and finally the hybridoma technology. Each of these methods has certain advantages and restrictions that determine their use for other purposes which is described elsewhere .The human hybridoma technology is a vital tool for production of human monoclonal antibodies. Early on, the possibility of using human mAbs for the prevention or treatment of human diseases was evident and was the driving force behind intensive effort to establish methods for human hybridoma. For this purpose, a selection of techniques could be used with varying efficiencies .One major obstacle to the development of human hybridomas over the years was the difficulty in collecting antigen-specific B cells to extend target populations. Antigen-specific memory B cells normally circulate in the peripheral blood at low concentrations, usually within a range centering about 1, or less, in 10,000 B cells. Therefore, it was difficult to generate human hybridoma cells, which would secrete desirable human mAbs. Recent technological developments in increasing the starting number of human antigen-specific B cells, improving the fusion efficiency and isolating new myeloma partners, and new cell cloning methods have allowed the development of protocols that make it possible to isolate B cells from blood samples and develop mAbs . Despite technical improvements in human hybridoma technology, there are no available human hybridoma cell lines developed against SARS-CoV-2. The selection of a proper blood donor is a key to this process. First, there may be some ethical challenges and restrictions concerning the collection of B cells from individuals. Special precautions must be taken about the origins of B cells to ensure patients’ anonymity and privacy . Then, a high titer of serum antibodies does not guarantee a high number of relevant peripheral B cells, but indicates a higher chance of effective mAb development .The use of unstimulated human peripheral blood lymphocytes (B lymphocytes) in mAb production is quite rare. On the other hand, human myeloma and lymphoblastoid cell lines could allow mAb production by means of fusion. Now we know how to increase the efficiency of fusion and consequently the efficiency of mAb production significantly by means of B-cell stimulation.Today, there is no longer any technical limitation to making human mAbs in the broadest sense. Biological problems involving the determination of the type and nature of any synthetic or natural antibody, the advantages of different immunological compartments of B cells, and various assays for the qualification and quantification of mAbs have been extensively solved .The most critical and challenging step in manufacturing human hybridomas is the fusion of desired lymphocyte populations with a myeloma partner effectively. There are 3 basic techniques used in mAb production to generate hybridomas: the use of (i) chemical agents such as PEG, (ii) fusogenic viruses, and (iii) electrical cytofusion. The most popular method used to generate hybridomas takes advantage of PEG’s fusogenic properties, while electric cytofusion is the most effective method of generating cell fusion and hybridoma . There are several myeloma cell lines that are suitable for fusion with human B cells and are available in the American Type Culture Collection (ATCC). Two of those cell lines, SHM-D3327 and HMMA 2.5, are frequently used for development of human mAbs .The final step in human hybridoma generation is the isolation of successful fusion products in the form of single-cell clones, which is a method also referred to as biological cloning. . There are now many approaches that can be used for biological cloning of human hybridomas. Traditionally, this was done by limiting dilution plating. More recently, advances in automated single-cell flow cytometric sorting, with indexing capabilities, have allowed fast, accurate and versatile single-cell plating. Finally, semisolid medium preparations can be used to grow single hybridoma cells as isolated, suspended colonies. This process can be highly automated with special clone picking devices and can also be carried out in an antigen-specific and semiquantitative fashion for the selection and biological cloning of high-producing human hybridomas .Phage display technology was discovered in 1985 by the 2018 Nobel prize laureate George P. Smith. He successfully integrated a foreign DNA into a filamentous M13 phage genome such that he could display the gene product on the surface of the phage . Today, M13 phage has been widely used in phage display technology applications because of its ease of use in the laboratory. It is a virus infecting specifically E. coli bacteria. M13 phage also differs from other phages by its unusual mechanism of producing progeny by continually releasing new phages from the bacterium without killing it . These 2 advantages made the M13 phage based display technology step forward amongst other phage display technologies.The phage display technology is based on the integration of a gene encoding a peptide or a protein fused with the phage coat proteins. The most extensively used coat proteins for display are the PVIII and PIII proteins; however, other coat proteins have also been used for display . Because of its high copy number (-2700 copies), the PVIII protein has been only used for the display of small peptides due to conformational problems hampering capsid formation . The PIII system, on the other hand, with its low copy number (5 copies), allows the display of larger molecules such as recombinant antibodies. The first phage display system displaying antibodies was described by McCafferty et al. (1990). They successfully displayed antibody variable regions on phages by using immunoglobulin variable genes of hybridomas and B cells. Since then, phage display technology has been extensively used for the discovery of antibodies or peptides against a large variety of antigens in many fields of application such as toxiconology , drug discovery , immunization , epitope mapping  and virus or toxin neutralization  by using phage peptide and antibody libraries.Antibody libraries have been displayed on phages in very different antibody formats. The main ones consist of Fab domain of antibodies, single chain Fv (scFv) which is the linear form of the variable domain of heavy and light chains, and single domain antibodies (nanobodies). The latter includes camelid VH domain and shark vNAR (new antigen receptor) domain . There are 4 types of antibody phage libraries, naive library, semisynthetic, synthetic and immune libraries . Naive libraries are generated from the natural antibody repertoire of donors or nonimmunized animals. Semisynthetic and synthetic libraries are generated from low diversity natural antibody repertoires by increasing the diversity with mutations in the CDR (complementarity determining region). Lastly, the immune library is generated from the antibody repertoire of immunized animals  or diseased or vaccinated humans . The selection of a target specific antibody from the phage libraries is made with a method called biopanning or affinity selection. The method consists of exposing the antibody phage library to the target antigen which is immobilized on a solid surface. The phages displaying antibodies specific to the target, bind to the antigen and the nonbinders are washed away from the media. Then the target specific phages are recovered by elution for a phage amplification step by infecting fresh bacterial cells. The phages are collected for a second round of biopanning. Generally, 3–4 biopanning cycles are sufficient to select antigen-binding antibodies .Since the COVID-19 pandemic emerged in December 2019, phage display technology has been intensively used for the development of neutralizing antibodies. Many different antibody libraries of different formats and strategies have been screened against SARS-CoV-2 spike protein and its receptor binding domain (RBD). Some of the studies have focused on screening previously developed libraries against SARS-CoV and MERS-CoV and finding cross-reactive antibodies. Others have performed screenings against semisynthetic or synthetic antibody libraries. Wrapp et al. (2020) reported that a previously developed a phage displayed single-domain antibody from llama, neutralizing the S antigen of SARS-CoV was also neutralizing the S antigen of the pseudotyped virus SARS-CoV-2 as a bivalent human IgG Fc-fusion protein. Zeng et al. 2020, constructed a phage displayed synthetic human Fab library (with an estimated size of 1 × 1012). The library was screened against the RBD domain of the SARS-CoV-2 spike antigen. They selected 2 antibodies with high affinity to RBD, however, only 1 showed neutralizing effect in competitive/blocking ELISA and pseudotyped virus neutralization assay. Wu et al. (2020) developed a phage-displayed single-domain antibody library by grafting naive CDR regions into the framework region of an allele in the human antibody heavy chain variable region. They made affinity selection against the RBD domain and the S1 subunit of SARS-CoV-2 and chose several neutralizing antibodies, including a “cryptic” epitope located in the spike’s trimeric interface. Liu et al. 2020, performed site-directed screening in a naive human scFv antibody library and domain antibody library by phage display against SARS-CoV-2 RBD. After several rounds of screening, they obtained 9 enriched clones from the domain antibody library and a single clone from the scFv antibody library. The scFv clone was reformatted into a human IgG1 antibody, while the domain antibody clones were fused with human Fc tag. By this way, Liu et al. revealed a potential neutralizing effect of these recombinant antibody structures with pseudotyped virus neutralization assay.On the other hand, with the increasing number of convalescent plasma uses from COVID-19 patients, the B lymphocytes have become readily available for the development of phage displayed human scFv and Fab antibody libraries. To this end, we have initiated the development of phage displayed human scFv libraries generated from convalescent plasmas of COVID-19 patients.The basis of display technologies is the use of genotype-phenotype relations. These technologies frequently use microbial systems like phage, bacteria, and yeast. In recent years, mammalian cell display technology has been introduced with some advantages such as improved efficiency in protein folding and posttranslational modifications. Compared to other display technologies, mammalian cell display technology comes to the fore by allowing the scanning of functional antibody structures and making use of FACS and in situ scanning methods. The highlight is that the library size that can be used for scanning is smaller than that of other systems (˂109) .Ho et al. (2006), transferred single chain variable fragments (scFv) of an antibody through transient (temporary) transfection to human embryonic kidney cells and expressed them on the cell surface. ScFv DNA was collected and analyzed using FACS  until scFv clones with the desired specificity were identified for the selection of the original antibody to the target antigen . In the report, they showed that all IgG antibodies could temporarily be produced on the cell surface and cells that synthesize antibodies against the target antigen could be selected through selection cycles based on FACS . Ho and Pastan (2019), announced that scFvs could also be transferred to the mammalian cell surface for affinity maturation. In the abstract of the report, the strategy for isolating high-affinity scFv specific to the CD22 antigen was identified as “mammalian cell display”. The strategy they have formed consists of the transient expression of antibodies on 293 T (HEK-293T) human embryonic kidney cell surface allowing clone selection by flow cytometry .Zhou et al., 2010, reported the display of full size IgGs on CHO cell surface. An important feature of the developed library was that recombinant DNA transfection was performed to include a single antibody replica gene in the genome of each cell. The use of the gene integration zone allowed the comparison of clones by a signal point. Along with the FACS system, this function proved to be capable of screening clones with high affinity and a high expression rate. Mammalian cell screening technology can also be used for antibody-based drug development for COVID-19 treatment. For this purpose, VH and VL antibody libraries can be generated with the blood samples of COVID-19 patients. Then, the libraries generated can be screened in mammalian cells against COVID-19 antigens . Up to our knowledge mammalian cell screening hasn’t been applied for the development of NAbs for SARS-Cov-2. Once NAbs are developed, it is vital to test them for their neutralizing efficiency. In other words, it is crucial to show that the virus, which has the power to create an infection, is effectively eliminated/neutralized by in vitro and in vivo systems. To this end, easy and safe screening methods –like ELISA and surface plasmon resonance (SPR)– could be helpful in decreasing the number of NAbs to be tested in neutralization assays. These screening methods would give an idea about the blocking capability of the NAbs on the viral spike protein and ACE2 interaction. Thus, the neutralization assays would be concentrated on the most potent NAbs.Plate-reduction neutralization test is used to titer neutralizing antibody for a virus. The serum or antibody to be screened is diluted and combined with a viral suspension. The antibody is incubated with the isolated virus and then transferred on host adhering cells. The surface of the cell layer is coated with a layer of agar or carboxymethyl cellulose to prevent the indiscriminate spread of the virus . The concentration of plaque-forming units can be estimated by the number of plaques (regions of infected cells) formed after a few days. Depending on the virus, the plate builder units may be determined through microscopic examination, or with specific dyes that react with fluorescent antibodies or infected cells. The concentration of serum indicates how many antibodies are detected or how effective it is in the reduction of the number of plaques by 50% compared to the serum-free virus. This calculation is defined as the value of PRNT50.Currently, PRNT50 is considered to be the “gold standard” to detect and measure antibodies that can neutralize viruses. It has a higher sensitivity than other tests, such as hemagglutination and many commercial Enzyme immunoassays. It is also more specific for the diagnosis of some arboviruses than other serological methods .One problem with this recently defined test is that the neutralizing ability of antibodies depends on virion maturation state and the type of cell used in the test. Therefore, if the wrong cell line is used for the analysis, the antibodies may appear to be effective in neutralization even if they are not in fact, or vice versa.Virus neutralization assay is used in combination with an infection experiment, such as PRNT. This assay detects a neutralizing antibody that can stop virus replication. Sera or culture supernatants are diluted and mixed with the virus . The mixtures are incubated approximately for 45 minutes at room temperature, and then the mixture is added on suitable cell lines  and the cells are incubated for 3 days at 37 °C .In a standard CPE (cytopathic effects) assay, a monolayer of cells is infected with a virus and then monitored for several days (or weeks) of morphological changes. The changes are expressed in distinct points corresponding to the sites of infection. Plaque assay has been the method of choice for several decades, where, shortly after the infection, the monolayer of cells is overlaid with semisolid material such as agarose. There are some variations in the plaque assays , however, all of them are labour-intensive, human dependent and hands-on jobs; thus, the determination of CPE is difficult. Therefore, it would be helpful to opt for using a more efficient and higher throughput alternative to traditional CPE assays: a real-time cell analysis (RTCA) system which is based on electric cell-substrate impedance sensing technology that can monitor cytopathic effects.In vaccine and drug research, preclinical studies must be conducted on animals. The efficacy of a candidate vaccine or drug is investigated first on mice, and then gradually on larger animals. The most critical stage on the way to a product is in vivo challenge experiments. To this end, the candidate molecule is given to mice with target microorganism and its effectiveness is tested. During the global fight with SARS-CoV-2, vaccine and drug development studies have gained a momentum. However, there are not much in vivo results for this virus in the literature yet. On the other hand, model animal studies were conducted on MERS-CoV and SARS-CoV diseases that caused infections in the past years. Therefore, in vivo studies of these viruses from the same family as SARS-CoV-2 will constitute a reference for new studies.Unfortunately, common small laboratory animals such as mice, ferrets, guinea pigs and hamsters were found not susceptible to MERS-CoV infection; because MERS-CoV receptor dipeptidyl peptidase-4 (DPP4) is a multifunctional transmembrane endopeptidase that breaks down insulin and other peptide hormones. For this reason, hDPP4 transgenic mouse model was created with C57BL/6 mice for MERS-CoV infection .Studies revealed that several inbred species of the mouse  promote replication of SARS-CoV, creating clinical symptoms of pneumonitis (129S) and SARS (aged BALB/C) .In mice immunized with SARS-CoV, the peaks of the virus titer in lungs occur at days 2–3 postinjection. However, at days 5–7 postinjection, the virus is cleared in most mice . In young mice, replication of SARS-CoV is not specifically associated with clinical symptoms and pathology of the disease. However, 129S6 mice were found to be more susceptible to SARS-CoV infection than BALB/C or B6 mice, with weight loss and early onset of pneumonia .In BALB/C mice, old ones experience a more severe disease, which results in increased mortality, than young ones, just like the case in SARS-CoV infection in humans . Therefore, being susceptible to age-related factors in diseases, aged BALB/C mice might also be an ideal animal model for SARS-CoV studies. The clinical symptoms of BALB/C mice include weight loss, dehydration, fur structure deterioration and histopathological damages such as viral replication in respiratory tissues and pneumonia .Roberts et al. (2008) investigated the susceptibility of various types of aged mice to SARS-CoV infection. They reported significant weight losses by aged B6, 129S6 (12–14 months) and BALB/C mice at days 3–5 postinjection being immunized with 105 TCID50 SARS-CoV (Urbani strain) . The same study also revealed similar results in terms of viral replication levels and kinetics in the lungs of aged B6 and BALB/C mice. At days 5 and 6 postinjection, B6 mice infected with SARS-CoV were observed to have 10–70 times lower virus titers than BALB/C mice .The referred publications indicate that the mouse models for SARS with these animals, except for aged or immunocompromised ones, did not develop significant clinical symptoms or reasonable mortality rates. For this reason, in recent studies, a new transgenic mouse model has been introduced to mimic human disease, to conduct pathogenesis studies and to develop antiviral treatments for SARS: K18-hACE2 transgenic mice, which express human ACE2, the receptor used by SARS-CoV . It has been shown that the K18-hACE2 transgenic mouse model may also be useful in studies on the outbreak and pathogenesis of the disease by novel coronavirus-2019 (SARS-CoV-2). In this context, Linlin et al. (2020) have studied hACE2 transgenic and wild-type (WT) mice to investigate SARS-CoV-2 virus pathogenicity.Challenge studies should be done carefully in animal bioSafety level-3 (ABSL-3) laboratories with personal protective equipment. To conduct a challenge study, the median lethal dose (LD50) of the infectious agent to be resisted must be known. Although Day et al. (2009) conducted studies on the median lethal dose in BALB/C mice using various SARS-CoV virus strains; regarding SARS-CoV-2, no literature is available on such studies, yet.Therefore, as a precursor of the median lethal dose, in vitro studies should be started to determine the median tissue culture infectious dose (TCID50). Day et al. (2009) studied 3 different SARS-CoV virus strains  with 3 different  median tissue culture infection doses (TCID50) in BALB/C mice. They observed the effects of these strains with different virulence factors on young and aged BALB/C through a 21-day challenge experiment. The survival rates of the animals per infection dose they received at different postinjection times and survival between the days of the defect were determined, and the virus titers in the lungs of the sacrificed animals were compared . These studies revealed that the hypotheses on the median tissue culture infection doses  were valid for SARS-CoV . Even if these doses are already determined, it is needed to conduct similar studies on animals to better calculate the virulence factor in terms of SARS-CoV-2.The golden Syrian hamster is an excellent model for SARS-CoV infection because viral replication is accompanied by pathological changes in the lungs including pneumonitis . Following intranasal inoculation with 103 TCID50 SARS-CoV, hamsters support viral replication in the nasal turbinates and lungs. Viremia is detected at days 2-3 post-injection, and the virus can be recovered from the spleen and liver . Although the disease produces histopathological symptoms in hamsters, clinical symptoms are not evident. The most striking example of this is reduced night movements in 5-10-week-old hamsters infected with SARS-CoV.Another disease that causes lung infection is influenza (H1N1). The model animal used in the vaccine studies of this disease is ferret. Studies have been conducted with these animals for SARS-CoV infection, which has the same target organ destruction.It has been demonstrated that Ferrets support SARS-CoV replication and develop multifocal pulmonary lesions that contain 5%–10% of the lungs’ surface area . It was determined that populations immunized with 103 or 104 TCID50 SARS-CoV caused virus replication in the lungs at 106 TCID50/mL on the fourth day . Compared to other vaccine studies for SARS-CoV, this model revealed poor efficacy. Therefore, more studies were needed to understand its immunogenicity . Nonhuman primates (NHPs) support viral replication and pneumonitis with variable clinical symptoms and pathology, depending upon the species. Various strains of SARS-CoV were tested on rhesus macaques , cynomolgus macaques , common marmosets , African green monkeys , squirrel monkeys and mustached tamarins. Studies revealed that squirrel monkeys and mustached tamarins are not susceptible to SARS-CoV Urbani infection. Among nonhuman primate models, rhesus and cynomolgus macaques were found to be the best NHPs in SARS-CoV studies.Sinovac Biotech Ltd. , a biotechnology company, has conducted a study on the protection of macaque monkeys from infection by SARS-CoV-2 by a vaccine candidate and announced that they attained a positive result. It is sure that the number of NHP studies will increase in vaccine development studies against the novel coronavirus-2019 (SARS-CoV-2) pandemic.Since the first detection of the novel coronavirus-2019 (SARS-CoV-2) in the Wuhan City of China in December 2019, researchers from all over the world are investigating and collaborating for the determination of the virus structure and its mode of infection in order to find a treatment against COVID-19 and a vaccine for a longer protection. The experience from previous virus epidemics indicate that cocktail of neutralizing antibodies are promising for a stronger virus neutralization. Therefore, it is important to discover several antibodies able to block the virus, and this variety can be achieved by the use of various antibody development technologies.Cocktail of NAbs has displayed a stronger neutralization than alone in treating both Ebola and SARS viruses. The generation of NAbs targeting various epitopes on SARS-CoV-2 would indeed be very important. In this way, combining a number of potent NAbs might reduce the possibility of the emergence of mutant strains.This work was initiated with the support of the Scientific and Technological Research Council of Turkey (TÜBİTAK Grant T1004-18AG020). Special thanks are extended to Melis Denizci Öncü and Filiz Kaya for their contributions to schematic representations and to Kemal Ulusoy for his proofreading.


New analytic bending, buckling, and free vibration solutions of rectangular nanoplates with combinations of clamped and simply supported edges are obtained by an up-to-date symplectic superposition method. The problems are reformulated in the Hamiltonian system and symplectic space, where the mathematical solution framework involves the construction of symplectic eigenvalue problems and symplectic eigen expansion. The analytic symplectic solutions are derived for several elaborated fundamental subproblems, the superposition of which yields the final analytic solutions. Besides Lévy-type solutions, non-Lévy-type solutions are also obtained, which cannot be achieved by conventional analytic methods. Comprehensive numerical results can provide benchmarks for other solution methods. One of the well-accepted models is the non-local continuum theory by Eringen10, which assumes that the stress at a point is a function of the strains at all the other points in the domain. Lu et al.11 established the non-local elastic plate theories based on Eringen’s theory, where the basic equations for the non-local Kirchhoff and Mindlin plate theories were derived, and the bending and free vibration problems of a rectangular nanoplate with simply supported edges were solved. It was shown that, for very small-sized plates, the influences of non-local effects on the mechanical properties are considerable.Nanoplates play an important role in the micro- and nano-scale technology, with applications to nano sensors, resonators, storage components, micro switches, etc. Mechanical behaviors, including bending, buckling, and free vibration, are frequently encountered in response to external excitations during the use of nanoplates. Accordingly, investigations on such behaviors are crucial for understanding the mechanical properties as well as providing guidelines for structural safety designs of relevant devices. In order to avoid enormous computational efforts when carrying out discrete atomistic or molecular dynamics simulation12 explored the small-scale effect on the buckling analysis of biaxially compressed simply supported single-layered graphene sheets (SLGS) by computing the buckling loads using the differential quadrature method (DQM), which was also employed for vibration analysis of SLGS embedded in elastic medium13. Malekzadeh and Shojaee14 extended a two-variable refined plate theory for the free vibration of nanoplates, where different types of boundary conditions (BCs) were studied by the DQM. Farajpour et al.16 used the DQM to analyze the buckling of higher-order and lower-order nonlocal strain gradient theory based orthotropic micro/nanoscale plates. Mohammadsalehi et al.17 investigated the vibration features of rectangular viscoelastic nanoplates with variable thickness by the DQM. Ghadiri et al.18 applied the generalized DQM to investigate the thermo-mechanical vibration of orthotropic cantilever and propped cantilever nanoplates. The method was also adopted by Ebrahimi et al.19 to analyze the thermo-mechanical vibration of rotating nonlocal nanoplates. Phadikar and Pradhan20 reported finite element formulations for nonlocal elastic Euler–Bernoulli beam and Kirchhoff plate, and analyzed bending, vibration, and buckling of simply supported nonlocal plates. Bahu and Patel21 developed an improved quadrilateral finite element for nonlinear second-order strain gradient elastic Kirchhoff plates based on the nonlocal theory. Necira22 developed the hierarchical finite element method for size-dependent free vibration analysis of Mindlin nano-plates with curvilinear plan-forms. Akgöz and Civalek23 employed the discrete singular convolution method for the free vibration and bending analysis of nano-scaled graphene sheets having sector shape. Babaei and Shahidi24 investigated the buckling behavior of quadrilateral SLGS under bi-axial compression by the Galerkin method, where the buckling loads of nanoplates with different geometrical parameters were obtained. Rahimi et al.25 studied the thermoelastic damping of in-plane vibration of the functionally graded nanoplates using the Galerkin method. Based on three-dimensional nonlocal elasticity theory, Shahrbabaki26 developed novel trigonometric series to be used as approximating functions in the Galerkin based approach in dealing with free vibration problems of rectangular nanoplates. Anjomshoa27 adopted the Ritz functions for buckling analysis of embedded orthotropic circular and elliptical micro/nano-plates under uniform in-plate compression. Chacraverty and Behera28 took the Rayleigh–Ritz method with algebraic polynomial displacement function to solve the vibration problem of isotropic rectangular nanoplates subjected to different BCs. Analooei et al.29 addressed the buckling and vibration characteristics of isotropic and orthotropic nanoplates using the spline finite strip method (FSM). Sarrami-Foroushani and Azhari30 examined the vibration and buckling characteristics of single and multi-layered graphene sheets by the FSM. Wang et al.31 presented highly accurate solutions for free vibration and eigen buckling of rectangular nanoplates with the iterative separation-of-variable (iSOV) method. Thanh et al.35 conducted the bending, buckling, and vibration analyses of microplates via the isogeometric method with couple stress theory, and further extended the method to the thermal buckling and post-buckling analyses of functionally graded micro-plates with porosities.Many studies have been conducted on modeling two-dimensional plate-like structures using the nonlocal plate theories. Some notable progresses by numerical methods are briefly reviewed in the following. Pradhan and Murmu36 reformulated the third-order shear deformation plate theory using the nonlocal theory, and presented analytical solutions of bending and free vibration of a simply supported rectangular nanoplate. Aksencer and Aydogdu37 used Navier-type solution and Lévy-type solution for vibration and buckling of simply supported nanoplates and those with two opposite edges simply supported. Sumelka38 proposed fractional calculus as a new formulation to study the nonlocal Kirchhoff–Love plates, taking the case of simply supported plate as an illustrative example. Based on Reddy’s nonlocal third-order shear deformation plate theory, Hosseini-Hashemi et al.39 obtained Lévy-type solutions for buckling and vibration problems of rectangular nanoplates. Ilkhani et al.40 applied the wave propagation approach to determine the natural frequencies of rectangular nanoplates with two opposite edges simply supported. Jamalpoor et al.41 adopted the Navier approach to solve free vibration and biaxial buckling of double-magneto-electro-elastic nanoplate-systems subjected to initial external electric and magnetic potentials. Moradi-Dasjerdi et al.42 applied the Navier approach at the free vibration analysis of nanocomposite sandwich plates reinforced with CNT aggregates. Arefi et al.43 adopted the Navier-form solutions to analyze the free vibration of a sandwich nano-plate including FG core and piezoelectric face-sheets. Cornacchia et al.44 obtained the Navier solutions for vibration and buckling of Kirchhoff nanoplates using second-order strain gradient theory. Besides, Yang et al.45 utilized the Bessel functions to settle the bending problems of circular nanoplates under concentrated and uniform loads.Although various effective numerical methods have been developed to study the mechanical behaviors of nanoplates, it is still important to explore new analytic methods because they cannot only provide benchmark theoretical solutions of permanent interests, but can also explicitly capture the relationships among different mechanical quantities, thus can serve as useful tools for validation of numerical methods, rapid parameter analyses, and efficient structural designs. However, due to the recognized mathematical difficulties in solving the complex boundary-value problems of governing higher-order partial differential equations (PDEs), the applicability of conventional analytic methods is generally restricted to some specific cases such as Navier-type and Lévy-type rectangular nanoplates, i.e., those fully simply supported or with at least two parallel edges simply supported. Some representative studies in this regard are briefly reviewed here. Aghababaei and Reddy47, buckling48, and vibration50 problems. The solution procedure involves three main steps, i.e., converting an original problem into several elaborated subproblems, solving the subproblems within the Hamiltonian system by the symplectic approach, and superposition of the subproblems for the final solution. Specifically, the symplectic eigenvalue problems of a Hamiltonian matrix are introduced, followed by symplectic eigen expansion, to yield the analytic solutions of the subproblems, which are exclusive mathematical techniques in the symplectic space51 rather than in the traditional Euclidean space. However, since the governing equations of the nanoplate problems are much more complex than those of the classical plate problems, there has been almost no research on developing the symplectic superposition method for analytic modeling of similar issues. In the following, for the first time, the symplectic superposition method is extended to obtain the analytic bending, buckling, and free vibration solutions of rectangular nanoplates with all combinations of clamped and simply supported edges, including both Lévy-type and non-Lévy-type solutions. Comprehensive benchmark results are presented to show fast convergence and high accuracy of the present solutions by excellent agreement with those obtained by the finite element method (FEM) and other numerical methods in the open literature. The effects of the nonlocal parameter and plate dimensions on the mechanical behaviors of the nanoplates are well examined with the present analytic solutions. Some useful conclusions are drawn to reflect the small-scale effects that are not captured in classical theories.In recent years, we have proposed an analytic symplectic superposition method for mechanics problems of plates and shells based on classical theories, which proved to be widely applicable to bending10, the transformed differential constitutive equation27 isl, and an experimentally defined material constant, E and h is the thickness of the nanoplate. The moment component resultants are thusBased on the nonlocal theory by Eringen31x and y are the coordinate variables; w is the transverse deflection of the nanoplate; x and y directions, respectively; The nonlocal theory-based quadratic functional for a nanoplate within the domain mentclass2pt{minimw yieldsThe variation of the governing equation  about w x axis. By coordinate exchange, we have H is a Hamiltonian operator matrix satisfying I2; accordingly, Eq.  nanoplate resting on an elastic foundation is first solved, where zero rotation conditions should be satisfied at each edge. Therefore, the following equations hold:For a uniformly loaded CCCC nanoplate, substituting Eqs. , 44), ,  into Eq.For a CCCC nanoplate under concentrated load, the only differences are on the right-hand sides of Eqs. –50, whicfor Eqs. –50, respFor the bending problem, equating quations –50 for tquations –54 for tquations  and 45)\documentquations /43), For the nanoplates with any other combinations of clamped and simply supported edges, the solutions can be obtained by relaxation of BCs from the above derivations. By equating ting Eq. , we haveing Eqs.  and 50)\documentComprehensive numerical results of the nanoplates with combinations of clamped and simply supported edges are presented to confirm the validity of the developed method, and, more importantly, to provide benchmark solutions for future comparison.b = 10 nm, 3, Rayleigh–Ritz method28, and iSOV method31, respectively, confirming the validity of the adopted nonlocal theory and the present method. The parameters adopted are as follows54: 56, which is not considered for a 5 nm × 2.5 nm nanoplate in reference28 and thus in the present study for comparison purpose.The convergence study is carried out and the results are shown in Table entclass1pt{minimab = 10 nm are tabulated in Tables 57 based on the FEM are also presented in each table, which correspond to the classical thin plate theory and are valid for the cases with b/200 is taken. Satisfactory agreement between the present solutions and their counterparts by the FEM is observed, further validating the present method.To provide more comprehensive benchmark solutions, we have tabulated the bending, buckling, and free vibration solutions of CCCC, CCCS, CCSS, SCSC, SCSS, and SSSS nanoplates in Tables 61. Accordingly, quantitative parameter analyses are implemented with the analytic solutions obtained in this study. Defining the critical buckling load ratio as the ratio of the nonlocal theory- to classical theory-based critical buckling loads, Fig. Figuring out the effects of the nonlocal parameter and others on the mechanical behavior of a nanoplate is helpful for researchers to determine the nonlocal parameter and to design the structureentclass1pt{minimaDefining the frequency ratio as that of the nonlocal theory- to classical theory-based frequencies, Fig. entclass1pt{minimaWith an up-to-date symplectic superposition method, the analytic bending, buckling, and free vibration solutions of rectangular nanoplates with combinations of clamped and simply supported edges are obtained based on Kirchhoff plate theory and Eringen’s nonlocal theory. Compared with conventional analytic methods such as the semi-inverse methods, the present method describes the problems in the Hamiltonian system, and yields the analytic solutions by the mathematical techniques in the symplectic space in a rigorous step-by-step way, without predetermining solution forms, which enables one to seek new analytic solutions. After validation of the present method by the other methods, comprehensive benchmark results are presented for both Lévy-type and non-Lévy-type nanoplates, including the bending deflections, critical buckling loads, and natural frequencies. Quantitative parameter analyses are implemented with the analytic solutions to gain insight into the behaviors and to provide reference for structural designs of nanoplates. It should be noted that the present work studies linear free vibration since the adopted superposition technique is applicable in linear regime with small deformation, but the studies on nonlinear vibration are definitely worthy of further exploration, which may constitute our follow-up work.


The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant–virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and  RNA silencing is a sequence-specific RNA degradation and inactivation mechanism, operative in most eukaryotes. It has also been implicated in the epigenetic events resulting in suppression of repetitive sequences including transposable elements (TEs) and imprinted genes. RNA silencing is now used as an umbrella term to encompass suppression of gene expression by all kinds of 21 to 24-nucleotide (nt) small RNAs (sRNAs), generated primarily due to the activity of enzymes like Dicers or Dicer-like proteins (DCLs). The sRNAs can be broadly categorized as small interfering RNAs (siRNAs) and microRNAs (miRNAs) in plant systems. This is grouped based on the mechanism of their biogenesis from precursor double-stranded RNAs (dsRNAs) or hairpin RNAs, respectively. The functional sRNAs are incorporated in the argonaute (AGO) protein of the RNA-induced silencing complex (RISC) that can act as a site-specific endonuclease on the cytoplasmic transcripts to enable posttranscriptional gene silencing (PTGS). The sRNAs can also target the genomic DNA in the nucleus to facilitate transcriptional gene silencing (TGS).Nia) transgene. This caused cosuppression of endo-Nia gene, resulting in lack of nitrogen availability and thus chlorosis . The spreading component involves intermediary steps of siRNA primed amplification or expansion of the silencing signals mediated by RNA-dependent RNA polymerases (RDRs or RdRPs). The spread of silencing was initially discovered by Palaqui and colleagues in transgenic tobacco bearing the nitrate reductase , as discussed below.The DCL2, DCL3, and DCL4 are involved in production of 22-, 24-, and 21-nt siRNAs, respectively . The siRThe DCL3 processes 24-nt siRNAs from transcripts that are initially transcribed from heterochromatic loci by RNA polymerase IV (Pol-IV) and then converted to dsRNA by RDR2 . The 24-One of the amazing characteristics of the silencing process is the requirement of catalytic amount of the trigger molecules that can cause silencing of numerous homologous transcripts . The silThus, the phenomenon of siRNA-mediated gene silencing involves both primary silencing and secondary or transitive silencing. In primary silencing, the trigger is an aberrant dsRNA that is processed into primary siRNAs mainly by DCL4. In transitive silencing, ssRNA templates are primed by primary siRNAs and are acted upon by RDR6 and SGS3 to produce dsRNA, which are processed by DCL2 or DCL4 to produce 22- or 21-nt transitive siRNAs, respectively , that deqde-1 gene of Neurospora crassa  oxidase (aco1) gene carrying an inverted repeat (IR) of the 5″ untranslated region (UTR) showed cosuppression of the transgene and endogenous aco1  or 3′ (P) of gfp, showed SS of the integrated gfp. These plants also exhibited silencing of nonoverlapping gfp sequences that were independently expressed from a potato virus X (PVX) virus–based vector  and the deletion alleles. The abundance of these secondary siRNAs appeared to decrease as a function of the distance from the primary trigger. The presence of the primary target mRNA was essential for the transitive effect, and it targeted sequences located 5′ to the mRNA sequences homologous to the primary dsRNA (The term ng dsRNA . In an ery dsRNA .gfp)-lacZ fusion construct, and the secondary target was a mitochondrially targeted gfp. Worms carrying both transgenes, when injected with dsRNA segments from lacZ, showed reduction of both nuclear gfp-lacZ and mitochondrial gfp. It was observed that a trigger that was located 3′ to the gfp-lacZ junction was most potent in the assay. Finally, To test whether these secondary siRNAs are capable of targeting degradation of homologous mRNAs, a transitive RNA-silencing assay was carried out by using two populations of target RNA . The priDrosophila, RDR primed with a synthetic 21-nt siRNA led to robust synthesis of a dsRNA of 690 bp in length in the embryo extracts  of which RDR1, RDR2, and RDR6 share a typical DLDGD signature motif sequence at the C-terminal catalytic site, and they belong to RDRα clade, whereas RDR3, RDR4, and RDR5 share an atypical DFDGD motif at the catalytic site, and they belong to RDRγ clade  infection, suggesting its role in the antiviral silencing  generation and cytosine methylation of the begomovirus genome. However, these plants were susceptible to RNA virus-like CMV (Ty1/Ty3 locus may be involved in siRNA amplification required for TGS (specific for DNA genomes) rather than the PTGS pathway.The function of members of the RDRγ clade remains elusive, although there are few evidences suggesting their role in plant defense. The At-RDR3 expresses strongly at inflorescence apex, whereas At-RDR5 expresses uniformly in different parts of is plant . RDR3 frilencing . Tomato sistance . Tomato like CMV . This inrdr6 mutant suggested its role in the spreading and maintenance of the silencing  of gfp under the SUC2 promoter, such that movement of GFP silencing outside the phloem companion cell (CC) vasculature could be monitored. This transgene generated large amounts of both 21- and 24-nt siRNAs that complexed with AGO1 to cause PTGS of sense transgenes. These could also prime the aberrant transcripts to initiate a cascade of reactions in the RDR6 pathway to produce a host of secondary siRNAs. Some of the secondary siRNAs could be detected outside the incipient cells because of systemic spreading  in grafting experiments also exhibited reduced amounts of 24-nt siRNAs and DNA methylation, indicating its role in the production of the silencing signal , a meiosis-specific cell cycle factor, caused suppression of the gene in the anthers of the first flowers of the WT scions.The mobile nature of RNA silencing was first demonstrated by the identification of sRNAs in the phloem sap of cucurbits . It was m plants . TransgeSilencing can manifest spontaneously up to approximately 10–15 cells from the point of initiation to form a zone of silencing . In addiN. benthamiana and Arabidopsis  can also spread cell-to-cell. SR movement of miRNAs has been demonstrated in bidopsis . For insbidopsis . Similarormation .The mobility of tasiRNAs has also been demonstrated during development of leaf polarity and lateral root growth . The eviThe transmission of silencing signal, leading to the suppression of the targeted gene in recipient cells (or sink cells), which are far away from the silencing originating cells (or source cells), constitutes SS. The first indication for SS came from the observations on spontaneous yet gradual propagation of PTGS from a localized area on a single leaf to the whole plant . Onset oArabidopsis might occur through plasmodesmata and not through the phloem  and sulfur (SUL), respectively. The siRNAs were produced in the CC, whereas the chlorotic phenotype indicated SR spread of silencing  via RDR6 and DCL4, resulting in increased cell-to-cell spread of PTGS , having high affinity to single-stranded sRNAs was identified . HoweverMobile sRNAs transfer epigenetic changes in recipient tissues, through both PTGS and TGS pathways. PTGS-mediated establishment of silencing in the recipient tissues was reported by two independent studies , because2O2). Using an elegant screen of Arabidopsis mutants impaired in the movement of root-to-shoot silencing, but not the production or effectiveness of the RNA-silencing signal, it was shown that the rci3 gene, coding for H2O2, contributes to mobility of the silencing signal  that function by promoting cleavage of target transcripts . Their bThe 21-nt tasiRNAs are derived from non–protein-coding TAS transcripts that are capped as well as poly-adenylated and contain a binding site mostly for 22-nt miRNA. The tasiRNAs can methylate the TAS DNA but do not influence production of TAS transcripts .The miRNA cleavage products are stabilized by suppressor of gene silencing 3 (SGS3)  and convA. thaliana, eight tasiRNA-producing loci have been identified that fall into four TAS groups (TAS1–TAS4). Among these, the TAS3 loci seem to be conserved in land plants. The tasiRNA production from TAS1a, b, c and TAS2 RNAs is initiated by the 22-nt-long miR173::AGO1 RISC and from TAS4 by the 22-nt-long miR828::AGO1 RISC , as well as few others , whereas effects .The conserved TAS3-tasiRNAs control a wide spectrum of biology by targeting the ARFs. These are the transcription activators that mediate the control of developmental pathways by plant hormone auxin. The ta-siRNA-ARF module is also one among the most conserved sRNA-target regulatory pathways in plants being present in the simple organisms, such as liverworts and ferns, to monocots and eudicots . It is iArabidopsis, TAS3 can spawn at least nine tasiRNAs, and of these, two tasiRNAs target two ARFs, namely, ARF3 and ARF4. It is likely that other tasiRNAs might target other ARFs. This targeting of ARF3 determines the abaxial fate of Arabidopsis leaves. It was shown that the biogenesis of conserved but low abundant tasiRNAs targeting ARF3 is restricted to the adaxial side, by the localized expression of AGO7 and TAS3. This generates an ARF3 protein–deficient zones that marks the adaxial side. However, the processed tasiRNAs move from the adaxial side to the abaxial side of leaf lamina, thus creating a gradient of sRNAs and its target ARF3. As the targeting is incomplete, it still keeps ARF3 protein visible and detectable to pattern the abaxial determinant ,” targets not only its parent transcripts but also other unrelated ones to produce secondary phasiRNAs , cleaves MYB-90 (PAP2), MYB-75 (PAP1), and MYB-113. All of these targets are involved in anthocyanin accumulation pathway and initiation of trichome in Arabidopsis leaves. Thus, these tasiRNAs act as a negative regulator of trichome initiation , resulting in explosive posttranscriptional silencing and subsequent poor growth of plants  phasiRNAs are also generated from protein coding as well as other genes with the help of initiator miRNAs following mechanisms similar to biogenesis of tasiRNAs. In fact, tasiRNAs could be deemed as a subset of phasiRNAs. However, phasiRNAs act in-AS genes . AccordiAS genes . The phaAS genes , proper AS genes , defenseAS genes , and abiAS genes .Arabidopsis miR825-5p targets MIST1 gene in the sequence coding for a highly conserved functional amino acid motif (TIR2) within the TIR domain of the receptor. As a result, trans-acting phasiRNAs are generated that, in turn, down-regulate a wide network of TIR-NBS-LRR (TNL) genes. Regulation through MIST1 affects disease resistance against the bacterial pathogen Pseudomonas syringae  phasiRNAs. These sRNAs negatively control expressions of the R genes in normal (uninfected) conditions to avoid the associated fitness cost. The syringae . For bio miR2118 . Similar miR2118 , whereas miR2118 . It was chanisms . The phaThe phasiRNAs were identified in diverse set of dicot species but not in monocots such as rice, indicating presence of different mechanism of pathogen resistance in monocots. This may also be attributed to the differential diversification of NBS-LRR genes between dicots and monocots , as indiMedicago PPR transcripts spawn secondary siRNA cascades, which are generated by an initial cleavage by miR1509 , SGS3 in peaches and soybean (triggered by miR2118 in soybean), and AGO2 in peaches. Reports of DCL-derived 21-nt phasiRNAs from papaya, sweet orange, tomato, and tobacco; AGO-derived 21-nt phasiRNAs from A. thaliana, Arabidopsis lyrata, monkey flower, tomato, and sorghum; and RDR-derived 21-nt phasiRNAs from foxtail millet and Amborella trichopoda are available in literature . In Arabidopsis, 9% of the whole genome is devoted for making cis-NATS, and almost all eukaryotic genomes encode cis-NATS. Each component of cis-NATS is coregulated, and their transcriptions are either environmentally or plant-developmentally controlled  of such pairs form hybrids (H), which are termed as 3′-3′ H, 3′-5′ H, or enclosed H . Such pantrolled . The mec strands . Approxicis. In Arabidopsis, DCL1 regulates more siRNA-associated cis-NATS (23.5%) than total cis-NATS (7.9%). This fact is strongly suggestive of down-regulation of sRNA producing cis-NATS by DCL1-dependent nat-siRNAs. However, DCL3-dependent nat-siRNAs may not be directly involved in the expression regulation of the NAT transcripts  transcript and similar to radicle induced cell death one 5 (sro5) transcript. Salt treatment of plant induces sro5 mRNA, which forms dsRNA at its 3′ end with the constitutively expressed P5CDH transcript. The DCL-processed siRNAs, namely, nat-siRNA-sro5, is further amplified by RDR6 and Pol IV. This siRNA then directs the cleavage of P5CDH transcripts, resulting in reduction of proline degradation and increase in salinity tolerance (P. syringae DC3000 strain carrying an effector gene avrRpt2 induce the ATGB2 transcript from a GTP-binding protein gene that base pairs with the constitutively expressed antisense transcript of PPR-like protein (PPRL). The resulting siRNA, namely, nat-siRNAATGB2, the biogenesis of which is dependent on DCL1, RDR6, SGS3, and NRPD1 (the largest subunit of Pol IV), down-regulates PPRL in its turn. As PPRL is the negative regulator of RSP-mediated resistance, siRNAATGB2 derepresses the pathway of effector-triggered immunity  gene locus in petunia × hybrida can be found in all tissues except roots, thus allowing the sho gene to direct cytokinin synthesis in the correct root locations  or ate them . These sate them . The hcsnd nrpd2 . The RDRnd nrpd2  and hetend nrpd2 . The 24-nd nrpd2 .Arabidopsis  locus with a direct tandem repeat in its promoter. Presence of methylation results in the WT phenotype, whereas absence of methylation causes the expression of the locus, resulting in the dwarf phenotype. Another example comes from the epialleles of flowering locus T (FT) where methylation of two enhancers located 5 kb upstream and 1 kb downstream of the gene can repress FT expression and results in delayed flowering phenotype. The third example of flowering Wageningen (FWA) locus, which determines correct flowering time, has been widely worked upon. It has two tandem SINE3-like retroposon repeats at its promoter, and transcribing sequence remains highly CG methylated and transcriptionally inactive throughout the vegetative development. FWA gets activated only in female gametophyte and endosperm by maternal imprinting. The FWA locus also is active in FWA mutant where hypomethylation occurs in the promoter of locus, resulting in delayed flowering , turnip crinkle virus (TCV), and peanut clump virus, respectively  could strongly reduce the production of secondary siRNAs, whereas P15, P25 (PVX), and so on, could not affect secondary siRNA production  in the incipient cell and also get transported to systemic regions to cause silencing (PTGS) in the recipient cells. The mechanism of cell-autonomous RNA silencing (CARS) is well established, but the mechanism of non–cell-autonomous RNA silencing (non-CARS) is still emerging . In CARSd shoots . Besidesbidopsis . But in chanisms .The transitive siRNAs can also be used to silence the endogenes. Van Houdt and colleagues employed a three-tier XYZ transgenic system and showed that the expression of transcripts  bearing no homology to the silencing-inducing locus  can also be decreased dramatically via transitive RNA silencing . This phNicotiana and concluded that transitivity-mediated silencing can affect endogenes only to a limited extent, but transgenes can be silenced at will with much higher efficiency. Arabidopsis. This approach can be employed to target a subset of the transcriptome in order to identify genes responsible for a particular localized process, such as photosynthesis  genes produce a pool of siRNAs, which are transported to the fungus and inactivate the fungal genes responsible for pathogen development and colonization  mRNAs during infection  have been adequately described in literature, and these also have been extensively used for engineering agronomic traits in crops . Here, wIn this approach, the target transgene gene for silencing (say X) is placed upstream of an element bearing an IR of 3′ UTR having heterologous sequence. The IR element is transcriptionally fused with X gene. It is better that a splice-able intron is sandwiched between the repeats. This cassette is then introduced in a binary vector and made ready for transformation in a plant system bearing the target gene X as a resident one. When introduced in plant, siRNAs are generated from the IR using the plant dicing activities. These siRNAs are then extended toward the 5′ end of the X-transcripts making use of transitivity. The average processivity of such extension by RDR6 enzyme is approximately 750 nt. In this process, the dsRNAs of X-transcripts are generated, which in turn are diced further, eventually destroying the transcripts of both transgene and endogene X. This transitivity-based approach is an efficient silencer but is weaker in efficiency compared to the hp-RNA–based constructs.Arabidopsis, namely, ap1 , ettin (encoding ARF3), and ttg1 (encoding a WD40 repeat protein regulating trichome and root hair development) using the transitivity-based vector. They have used the IR of octopine synthase (ocs) terminator as a source of heterologous siRNAs in their transitivity vector. They have also found that hp-RNA vectors are superior to transitivity vectors in silencing the target genes , which consequently silence their targets in a usual manner using the RNA-silencing machinery of the host plants.These vectors generate secondary siRNAs directly and are not dependent on induction of primary siRNAs. The TAS1c locus in A. thaliana was engineered by de la Luz Gutierrez-Nava et al. to silence the FAD2 gene by replacing a single native tasiRNA with an siRNA targeting FAD2 (siFAD2) or replacing five native siRNAs with siFAD2. Such strategies showed silencing phenotypes akin to that of a fad-2-1 null mutant. Multiplexing of several atasiRNAs can also lead to simultaneous silencing of several related or unrelated genes. The construct retaining the binding site of miR173 but including a large part of gene of interest by removing the bulk of TAS1c sequences is also effective in inducing silencing. The atasiRNAs can be computationally designed with user-friendly web tools such as P-SAMS1 , EARLY FLOWERING 3 (ELF3), FLOWERING LOCUS T (FT), and LEAFY (LFY) and demonstrated that the MIGS constructs were capable of mimicking the phenotypes ag2, elf3-9, ft-10, and lfy-12 null mutants  and PDS gene-silencing phenotypes were achieved when the Arabidopsis miR173 precursors were cotransformed along with the MIGS constructs. Here the mature miR173 was processed in a manner different from Arabidopsis, and both phased and out-of-phase siRNAs resulted in the plant tissues. Silencing of the targeted endogenes was very efficient, but the molecular mechanisms of silencing between Arabidopsis and petunia plants were different  the possibility that MIGS-derived tasiRNAs can induce transitivity as reported  is placed under the control of such peculiar miRNAs and allowed to transcribe in plants, phased 21-nt siRNAs (phasiRNAs-like) are generated, and the silencing constructs harboring the mentioned elements are known as miRNA-induced gene silencing (MIGS) vectors. The MIGS term was first coined by de Felippes and colleagues when they used the 22-nt miR173 binding site upstream of many  mutants . When thifferent . Similaruncatula , soybeanuncatula , and ricuncatula . Despitereported . Finallyreported .Previously, we have seen that plants transport secondary siRNAs in invading pathogens and pests such as fungi, nematodes, insects, etc. Although the specific mechanism of transfer is not fully revealed, this principle can be exploited to develop antipathogen strategies. The siRNAs, which can specifically and crucially target developmental genes of pathogens, can be expressed in plants in a transgenic manner. When these siRNAs are passed on to the invading organisms, they inactivate the target pathogen genes using the RNA-silencing machineries of the pathogen, thus crippling the growth of pathogens.Meloidogyne incognita. The nematode secretes a small signaling peptide, namely, 16D10, in the plant cells that causes pathogenicity by promoting accelerated and enlarged root growth. Huang et al. expressed dsRNA as well as hp-RNA of 16D10 in Arabidopsis and challenged the roots of transgenic plant by M. incognita. As a result, the numbers of galls and M. incognita eggs on transgenic A. thaliana plants were greatly reduced because of reduction in secretion of peptide  is made recombinant with 258-bp-long sequence of the rust fungus, and subsequently common bean was allowed to infect with recombinant virus followed by challenge with the fungus. The 258-nt RNA contained five U. appendiculatus candidate effector mRNAs. Four of the five effectors targeted rust disease symptoms, and the BPMV-infected beans showed less rust disease symptoms and less of fungal RNA. The virus-based HIGS has also been applied to resist insects of various kinds and has been summarized well by A non-transgenic expression of dsRNAs targeted to pathogens and pests in plants can also be achieved through virus-based vectors. This approach may be called virus-induced HIGS and has been successfully used to silence plant pathogens and insect pests in a number of different plant species -free, virus-free procedures. Fortunately, plants internalize externally applied dsRNA and process those as siRNAs to effect silencing. Externally applied dsRNA, hp-RNA, siRNAs, etc., have been shown effective to cause silencing of endogenes and transgenes of plants, as well as genes of invading organisms such as viruses, fungi, insects, nematodes, etc. . The exoin vitro transcribed 685-bp dsRNA and/or chemically synthesized 21-nt sRNAs targeting the endogenous PDS on the leaves of N. benthamiana plants, which were pretreated with Silwet L-77 surfactant. As a result, the endogenous PDS mRNA displayed extensive PDS RNA silencing . This protein has been shown to inhibit antiviral silencing spread . PreventPotexvirus PVX and the potyvirus plum pox virus (PPV) as the vehicle of VIGS in the WT and rdr6-deficient N. benthamiana plants. The VIGS efficiency was seriously dampened in rdr6 mutant plants compared to the WT, although the accumulation of VsiRNA was as similar in rdr6 mutants as that in the WT. It was concluded that the high accumulation of primary siRNAs of PVX and PPV in rdr6 mutant plants was ineffective in causing VIGS-mediated silencing , a large number of PHAS loci showed substantial changes in PhasiRNAs following viral infections. The ARF gene in rice showed more than 8- and 3.5-fold changes in phasiRNA generation upon RSV and RDV infections, respectively. The authors suggested that viral infection-induced or -suppressed expression of selective phasiRNAs may act as a novel mechanism to regulate the expression of selective host genes  are also altered following virus infection. Some miRNAs are overexpressed, resulting in overproduction of PhasiRNAs, whereas in other cases, PhasiRNAs disappear altogether. Such alterations lead to fine-tuning of the host genes related to resistance, susceptibility, or tolerance toward the infecting virus . Zheng est genes . In anotst genes . Similarwith CMV .Abutilon plants from the infective variegation by a virus, which is known today as Abutilon mosaic virus (AbMV)  . Later os (AbMV) . Later ps (AbMV) . This phs (AbMV) . Until tin vitro and genetic analyses with the recovered leaves showed that PTGS-mediated degradation of viral mRNAs occurs frequently, resulting in lowering of viral titer in the recovered leaves. Both VsiRNAs and viral genomic RNAs exist in low quantum, and thus, the disease expression is minimized (or nil) in recovered leaves  or siRNA-dependent RdRM pathways. However, the Arabidopsis mutants of secondary VsiRNA-degrading enzymes, namely, xrn4-3 and ein-5, showed enhanced recovery, supporting the role of secondary VsiRNA in recovery. Both RDR2 and RDR6 along with SGS3 and DCL4 were very much necessary for recovery. Taking all observations together, it appears that recovery process establishes a state of tolerance in the infected tissues and occurs following robust mobility of antiviral secondary siRNAs from source to sink tissues. The recovery also establishes a sufficient dosage of VsiRNAs that can block the VSR activity involved in the formation of disease symptoms  is different, and here the meristem exclusion and recovery are quite independent of each other. The exclusion of PVX involves a silencing mechanism that initiates in lower uninfected tissues or systemically infected leaves and spreads ahead the viral infection front and depends on a long-range RNA-silencing signaling regulated by host RdRP RDR6  infecting N. benthamiana highlights an altogether different pathway. By carefully inactivating the antiviral silencing machinery, the Burgyan group showed that the virus cannot enter tobacco SAM at all, thereby implicating that RNA silencing is not behind the SAM exclusion of CymRSV. The authors investigated the transcriptional changes following virus infection in the shoot and were of the opinion that these changes are the key factors behind tip necrosis and symptom recovery. They also observed that glyceraldehyde 3-phosphate dehydrogenase failed to express in tissues around the meristem, and absence of this factor is the principal cause of meristem exclusion as this protein is required for virus replication and growth –dependent methyltransferases. SAM-methyltransferases are required for-RNA processing and ribosome stability. This finding also establishes WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants  need to be identified for efficient antiviral silencing. in vitro extract can dice the dsRNA of TBSV in three siRNA forms, but 24-nt siRNAs were the predominant species. The siRNAs were allowed to be incorporated in the flag-tagged AGO1 or AGO2 in presence of the TBY2 extract. The AGOs were in vitro translated and supplemented in the RISC-incorporation assay. The incorporated VsiRNAs were then detected from immune-precipitated RISC by NGS technique. A substantial portion of diced VsiRNAs was not found in RISC. Some of these RISC-incorporated siRNAs were finally selected, and their synthetic forms were programmed with RISC in a slicing assay prepared with the TBY2 extract, and again, only a few of the selected ones were competent for slicing. The slicing-competent siRNAs were the eVsiRNA as these can be used efficiently for resistance against TBSV  siRNAs come up that are highly dependent on host RDR1 protein for biogenesis. But these expressed siRNAs vanish altogether in presence of CMV-2b RNA-silencing suppressor. These mostly 21-nt siRNAs are called vasiRNAs, which are more like VsiRNAs and are produced predominantly by DCL4. For their biogenesis, they do not require SGS3 and RDR6, and these efficiently load on AGO2 complexes, whereas a small fraction also load on AGO1 complexes. The vasiRNAs target the expression of the host genes from where these are derived, and such down-regulation leads to antiviral silencing. The overall function of these vasiRNAs is to broaden the scope antiviral silencing. Various strains of CMV, which are deficient in 2b protein, are also competent to produce vasiRNAs. TuMV also generates vasiRNAs in Arabidopsis with similar genetic requirements such as CMV, but the viral RNA suppressor, i.e., HC-Pro, does not have any effect on the production of vasiRNAs. The extent of accumulation of vasiRNAs depends on the siRNA degrading factors such as exoribonuclease 4/ethylene-insensitive 5, and the loss of these factors enhances the level of vasiRNAs. Such enhancement also results in higher virus resistance without having any effect on VsiRNAs (When CMV with its 2b gene inactivated (CMVΔ2b) infects  VsiRNAs . A host  VsiRNAs . In rice VsiRNAs .Geminiviridae, Nanoviridae, and Caulimoviridae harbor DNA genomes. Of these, TGS of geminiviruses has been worked upon to some details, whereas the same for other family members are underreported. So, we would focus here on geminiviruses only. The single-stranded circular DNA of geminiviruses enter plant nuclei following infection that gets quickly converted to dsDNA forms known as replicative forms (RFs). DNA replication and transcription initiate with the RF templates. However, the RF templates do not remain in the naked form but complex with histones to form nucleosome-like structures. They look like a ring of 13–14 beads in electron microscopy, and biochemical assays reveal that the IR region of viral DNA and promoters of early transcripts such as Rep, AC3 etc., are somewhat free of histones , ago4, and ddm1, show hypersensitivity to CaLCuV and BCTV. Similar increased sensitivity is observed in mutant plants lacking the non-CG methyltransferases drm1/drm2 and cmt3 and the H3K9 methyltransferase kyp2/suvh4. Hyper susceptibility is also evidenced in adenosine kinase mutants (adk1 and adk2), which are interrupted in methyl cycle, providing Sam which is the key cofactor for methyltransferase. As complete loss of Met proteins results in growth defects of host plants, experiments with met1 mutants (CG maintenance methyltransferase) are difficult to carry out. However, met1 heterozygotes exhibit normal growth but show moderately enhanced susceptibility to the geminiviruses  beta-C1 interacts with S-adenosyl homocysteine hydrolase and blocks its activity in vitro ; and transgenic plum with resistance to PPV released in the United States (in mid 2000s) .Rep coding sequence. The transgenic plants overproducing 21- and 22-nt siRNAs have been found almost immune to TYLCV challenge  have been used successfully. We have shown that tomato transgenics overproducing the amiRNAs to silence the conserved regions of ToLCV- AV2/AC2 genes can very well tolerate various leaf curl viruses of tomato . EarlierN. benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each amiRNA construct is monitored by inspecting appearance of viral symptoms and carrying out molecular analyses of virus titer accumulation in infected tissues. This methodology has been used to identify highly effective amiRNAs against a viroid PSTVd and the virus tomato spotted wilt virus (TSWV) in N. benthamiana. A detailed account of various amiR constructs to confer virus resistance in plants has been given in many recent reviews  constructs has been described. These are WMD3 and P-SAMS, which are optimized for both effectiveness and specificity of designed amiRNAs. The web-predicted amiRNAs are transiently expressed individually in several  reviews .Two types of silencing tools have been used in literature that generates Syn-tasiRNAs/PhasiRNAs to silence genes of interest. These tools rely solely on the action of secondary siRNAs and have been used extensively for both basic studies of gene function and improving agronomic traits of crops. Here we would focus only on antiviral activities of these tools.Artificial or synthetic siRNAs (atasiRNA or syn-tasiRNA) are expressed in plants from engineered TAS sequences of Arabidopsis. A single or few tasiRNA sequences of precursor TAS DNA are removed and substituted by sequences of interest of equivalent sizes in the same position in the engineered TAS DNA. The engineered TAS DNA acts as a transgene and, when introduced in plants, gives rise to syn-tasiRNA primary transcript (pri-syn-tasiRNA), which eventually spawns phased syn-tasiRNA duplexes of 21 base pairs in the usual tasiRNA biogenesis pathway. When TAS1 precursor is used, miR173/AGO1 complex acts an initiating slicer. Similarly, miR390/AGO7 is the slicer when the precursor is TAS3a RNA. In all cases, the guide strands of syn-tasiRNAs are incorporated into AGO1/RISC, which functions as a site-specific endonuclease to degrade multiple sites of one or multiple viral RNAs as guided by syn-tasiRNA sequences. As such silencing processes of target genes are induced by synthetic tasiRNAs, these are also called SIGS. An account of these target genes can be found in several reviews , M (medium), and S  RNAs. The entire genome codes for six proteins via five different ORFs. The L component is completely antisense and encodes the RdRP; the M segment is ambisense and encodes the NSm MP and the structural Gn/Gc proteins required for vector transmission; the S component is ambisense and encodes the nucleocapsid N protein and the silencing suppressor NSs.Most of the contributions on antiviral strategies using SIGS have come from the lab of N. benthamiana and S. lycopersicum plants, respectively. As the atasiRNAs/syn-tasiRNAs are computationally designed with user-friendly web tools such as P-SAMS (see text footnote 1)  , the indN. benthamiana) a-miR sequences for silencing the various viral genes. Five a-miRs from each of the three genomic components were selected that matched with their TSs nearly perfectly and were distributed throughout the genome. All of the L-amiRs targeted RdRP mRNA, three and two M-amiRs targeted NSm viral RNA and mRNA respectively, and one and four S-amiRs targeted N-mRNA and NSs viral RNA, respectively. These sequences were then subjected to high-throughput functional analyses for their anti-TSWV activities in agroinfiltration-based transient assays in N. benthamiana. Of all these 15 amiRs, only two from L segment (amiR-TSWV-L3/L5) and other two from the M component (amiR-TSWV-M1/M3) were efficient in controlling TSWV local and systemic lesions in N. benthamiana. These four sequences were used to construct the SIGS vector using the TAS1c DNA backbone, and the synthetic tasiRNAs were expressed in N. benthamiana transiently using the 35S promoter. For expression of syn-tasiRNAs, coinfiltration with miR173 was necessary as the tobacco plant do not encode the miR173. A highly anti-TSWV response was observed with the SIGS vector. Finally, the investigators examined the comparative silencing activities of amiRs and syn-tasiRNAs and concluded that the syn-tasiRNAs were much better silencing tools than the a-miRs.As there are many TSWV isolates that escape the spell of resistance gene like SW5, Carbonell’s group came up with development of broad-range anti-TSWV strategies employing the SIGS technique . They usArabidopsis and tobacco, but Carbonell’s group established the same for the first time in a crop plant, namely, tomato, the natural host of TSWV. The aforementioned four amiRs and the syn-tasiRNAs were transgenically expressed in tomato independently. For syn-tasiRNA expression, the transgene also included the miR-173 expression cassette as the tomato plant does not encode this miRNA. Several transgenic lines with variable levels of expression of syn-tasiRNAs were obtained. When these lines were examined for anti-TSWV activities, it was found that low expressing two lines failed to show resistance, but in all others (10) transgenics expressing suitable amount of syn-tasiRNAs were highly resistant against challenge TSWV. In contrast, the majority of the amiRNA lines were susceptible, and only a couple of lines with higher amiRNA accumulation were resistant. A systematic analysis of the a-miR TSs in the progeny viruses from infected plant leaves revealed the emergence of TS mutations exclusively in susceptible amiRNA lines. The investigators concluded that subinhibitory amiRNA accumulation led to the emergence of TS mutations in replicating TSWV, whereas simultaneous multiplexed targeting of viral RNAs with several syn-tasiRNAs restricted the capability of escape mutations, thus establishing robust plant resistance  of the expressed syn-tasiRNAs and tweaking the sequences of the same . The invArabidopsis miRNA 173 (for TAS1) and miR390 (for TAS3), there is a host of miRNAs that are used for fine-tuning the regulation of plant development and immunity genes. A number of 22-nt miRNAs, such as miR482/2118, miR1507, miR2109, miR5300, and miR6019, trigger biosynthesis of phasiRNAs from the nucleotide-binding leucine-rich repeat (NB-LRR) gene family containing hundreds of members in eudicots that constitute the majority of plant disease resistance (R) genes. These phasiRNAs suppress the expression of these resistance genes to avoid toll on plant growth in absence of pathogen invasion  or phasiRNA generation [with mutation in RDR6 (rdr6) genes] exhibit enhanced effector-triggered immunity–based resistance to some pathogens . Besides the invasion . Arabidoathogens . The miRathogens . Similar  were sandwiched between 5′ and 3′ binding sites of miR390, a 21-nt miRNA which is mostly conserved in plant species and triggers tasiRNAs from TAS3 locus. In this construct, the whole of TAS3 DNA was removed and replaced by VSR AC2/AC4. When this construct was agroinfiltrated in tobacco or tomato, phasiRNAs of 21-nt size were generated in the infiltrated leaves. A central mutation spanning 4 nt in the 3′ binding element abolished the generation of siRNAs, establishing thereby that the siRNAs did not result from PTGS of the introduced construct. To check functionality of the tasiRNAs, the ones derived from the AC2 gene were allowed to act on viral transcripts that were made from introduction of the infectious viral clone in plants. The infectious viral clone was introduced on leaves that were at the upper side of tasiRNA generating leaf. The cleavage points on the viral transcripts were mapped by degradome analysis and were found to be localized only in the AC2 transcripts and nowhere else in the entire viral transcripts. This observation showed that the tasiRNAs are functional and mobile as well. We then carried out transient agroinfiltration assays to check resistance to ToLCNDV, and the vast majority of the plants showed resistance against the virus at the systemic leaves. Subsequently, we generated stable transgenics with these constructs in both tobacco and tomato. Approximately 95% of the plants showed features of tolerant plants against ToLCNDV and similar viruses. The virus resistance was monitored by inspecting reduction of leaf curl and stunting characteristics of the transgenics, as well as molecular analyses of viral DNA formation in the systemic leaves of T0 and T1 transgenic plants. A few of the transgenic lines were almost immune to the invading ToLCNDVs at the T0 and T1 stage , 2019.Arabidopsis endo- genes have been silenced using this approach. The atasiRNA/MIGS vectors produce siRNAs based on biogenesis of secondary siRNAs that can be utilized in plant development, flower control, and plant defense against viruses. Abiotic stress-resistant plants can also be developed using such vectors.RNA silencing in plants involves the movement of amplified 24-, 21-, 22-nt sRNAs for SR spread and SS, resulting in PTGS or TGS. The SR silencing spreads from cell-to-cell through the symplastic movement, whereas SS follows a phloem-based translocation pathway from (metabolic) source. In the last few years, we have witnessed substantial progress in interpreting the mechanisms of RNA-silencing spread. Such mechanisms are utilized to amplify siRNAs or transitive RNAs that have great roles to play in biotechnological work. Any gene of unknown sequence can be silenced if transitive RNA is allowed within the transcript of target gene. The secondary siRNAs can be used to silence any unrelated gene having no homology with the silencer siRNAs. Many C. elegans, secondary siRNAs are chemically different from their primary counterparts. The 5′ ends of secondary siRNAs have di/triphosphates, whereas primary siRNAs have monophosphates. So, we need to know if plant secondary siRNAs are chemically different from the primary ones. Careful chromatographic analyses coupled with the biochemical treatments of the pool of siRNAs can resolve this question. Primary siRNAs are protected from nucleolytic degradation by addition of sugar moieties by Hen1 proteins. Are the secondary siRNAs treated similarly by Hen1 enzymes? Even if they are, do they retain the sugar moieties during their intercellular trafficking? How do the secondary siRNAs move out of the incipient cells and move in distant recipient cells? Are there channel proteins that help them in such trafficking? Do these siRNAs acquire novel chemical tags during their journey across the cells as there could be kinases and phosphates and other nucleic acid–modifying enzymes residing on the cellular membranes? In the intracellular silencing, DCL4 plays the major role, but for intercellular silencing, DCL4 is inhibitory, and DCL2 plays the major role. So the question remains: How such distinctive regulation occurs, and what are the regulatory factors? It is often said that processes of intracellular silencing are well worked out, but it is not clear if similar mechanisms are at work for intercellular silencing. Does the intercellular silencing require different participatory factors? We anticipate that these unresolved questions will be uncovered soon in the future.Although the importance of secondary siRNAs for plant growth and defense has been appreciated in literature in recent years, there are lot of undiscovered, dark matters associated with their biogeneses and functions. In some organisms such as in vivo factors, which are difficult to predict by bioinformatics means. It has been established that not all VsiRNAs can cause silencing of viral genes. Not all VsiRNAs are incorporated in RISC complexes, and not even all of those that are incorporated in RISC can cause silencing. Hence, it is important to find the effective e-VsiRNAs that are surely capable of causing silencing. Fortunately, the procedures prescribed by 2, etc., can screen for desirable and effective siRNAs. Use of non-constitutive promoters  to express siRNAs can also cut down the quantum of off-target effects. The sequences of syn-tasiRNAs are derived from appropriate bioinformatics followed by experimental validation, and thus, these are relatively free of off-target effects. Moreover, these syn-tasiRNAs have the advantages of regulatable silencing, which is often required  might have cryptic secondary structures, or the site could be inaccessible due to preoccupancy by required . Thus, iNS-M and SM prepared the initial draft. All authors participated in extensive editing of the manuscript and read and approved the write-up.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.