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Abstract Application of exogenous doublestranded RNA （dsRNA） for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However， the instability of dsRNA is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only five days. Innovative approaches， such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release， are emerging as key disruptive technologies. Knowledge about the mechanism of entry， transport and processing of exogenously applied dsRNA in plants is still limited. In addition， cost of dsRNA and regulatory framework are still key influencers towards practical adoption of this technology.
Key words Plant; dsRNA; Induction; Virus; Resistance
With the development of biotechnology， an increasing research effort has been devoted to exploring the pest and disease resistance of crops in recent years. As a phenomenon inherent in living organisms， RNA silencing plays a decisive role in growth， development， and host defense against viral invasion. RNA silencing has been shown to be an effective way to protect plants against viruses， viroids， nematodes， pests and fungi[1-9]. Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. A target gene in a biological cell can be specifically silenced by inducing an exogenous homologous sequence， without influencing the expression of other genes. Gene silencing can occur during transcription （transcriptional gene silencing， TGS） or after transcription （posttranscriptional gene silencing， PTGS）[10-11].
Plant virus diseases can greatly reduce crop yield and affect local and global food security. The challenges faced by virus disease prevention and control include the lack of resistance genes， the emergence of new plant viruses， insecticide resistance of insects that spread viruses， and the implementation of disease control measures in farmland， as well as the lack of plant antiviral agents.
RNAmediated resistance to viruses is highly safe to the environment. No viral proteins can be synthesized in the cells of plants that have been transformed with untranslated genes or nucleotide sequences， which avoids the occurrence of heterologous encapsidation. These types of resistance， however， seem to be effective only against viral strains closely related to the source of the transgene. To endow the plants with resistance against multiple viral species， the effective nucleic acid sequences of several viruses are ligated together and transformed to plants. However， such method is expensive， time consuming， but also requires efficient protocols for plant transformation. In addition， genetically modified plants， especially genetically modified food crops have not been well accepted by the public. In order to overcome these limitations and public concerns about genetically modified food crops， dsRNA is being explored as a topical application to trigger the RNAi pathway against pathogenic viruses[12]. Viral RNA Silencing Induced by Exogenous Doublestranded RNA
Recent research reports have shown that RNAimediated viral silencing in plants can be achieved by introducing exogenous dsRNA[13-14]. Tenllado and DiazRuiz[14] reported that dsRNA derived from viral sequences could interfere with virus infection in a sequencespecific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacteriummediated transientexpression assay. For example， mechanical inoculation of pepper mild mottle virus （PMMoV） and dsRNA homologous to PMMoV replicase prevented the formation of virusinduced lesions on the inoculated leaves of Nicotiana tabacum cv. Xanthi or Capsicum chinense.
Tenllado and DiazRuiz[14] also reported that dsRNAinduced viral immunity is dependent on the sequence specificity and minimum homology length between the dsRNA and the target virus. For example， a 0.6-1.0 kb dsRNA fragment that is 100% homologous to PMMoV is capable of inhibiting viral infection， and a dsRNA fragment less than 300 bp in length or low in homology shows a reduced ability to inhibit viral infection.
The dsRNA fragments used to induce resistance against viruses were artificially synthetic in most initial studies. Then， it was found that a large amount of dsRNA can be produced using an RNase IIIdeficient strain HT115 of Escherichia coli[15-16]. Crude extracts of the bacterially expressed dsRNA can be used to protect plants against the infection of viruses such as PMMoV and PPV in tobaccos[17-18].
The application of dsRNA in crop disease control has drawn more and more attention since its first report[14， 18-20]. In the study of Gan et al.[21]， two fragments of the Sugarcane Mosaic Virus （SCMV） CP （coat protein） gene were cloned into the invertedrepeat cloning vector， and transformed individually into E. coli HT115， and then the crude extracts of E. coli HT115 containing large amounts of dsRNA were sprayed to plants， and the results demonstrated that spraying crude dsRNAcontaining extracts inhibited SCMV infection. In addition， this study also showed that spraying plants with SCMV CP1 dsRNA provided protective effect against viral infection for only five days， which was consistent with the findings of Tenllado[14].
However， the mechanisms of uptake and spread of dsRNA， and how they are converted into siRNAs are still poorly understood[22]. A recent study by Konakalla et al.[23] showed that exogenously applied dsRNA exhibits a rapid systemic trafficking in planta， and it is processed successfully by DICERlike proteins producing siRNAs. This study also showed the fast systemic spread of TMV p126 dsRNA from the treated （local） to nontreated （systemic） leaves beginning from 1 h postapplication， and stemloop RTPCR confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves postapplication. From all above studies， it can be concluded that systemic RNAi resistance against virus can be induced by sequencespecific dsRNA. However， the instability of dsRNA is still a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only five days.
Nanoparticlemediated Delivery of dsRNA
The instability of dsRNA sprayed onto plant leaves is still the biggest challenge in viral control using dsRNA. In the study of Mitter et al.[24]， a new layered double hydroxide （LDH） nanomaterial was used as a carrier for topical delivery of RNAi for sustained protection against plant viruses， which can not only improves the stability of dsRNA， but also result in slow release of dsRNA over several weeks.
LDH nanosheets which are nontoxic， biodegradable and biocompatible[25]， can load dsRNA to form dsRNALDH complexes referred to as BioClay[24]. After BioClay is sprayed onto plant leaves， it slowly degrades under the action of carbon dioxide and water in the atmosphere， and release dsRNA. The LDHcombined dsRNA cannot be easily eluted from plant leaves， and can provide protection against viruses for 30 days.
Previous studies[13， 24， 26] have shown that dsRNA can be taken up by plant cells and induce silencing of an endogenous gene. One explanation is that dsRNA is rapidly transported into other cells after being absorbed. Significantly， a single spray of dsRNA loaded on LDH （BioClay） afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves[24]. The mechanisms of action on the absorption， transport and processing of dsRNA are still poorly understood. Nanoparticlemediated RNAi in cancer prevention and treatment has become a research hotspot in recent years[27-28].
In the studies of Ladewig et al.[29] and Wong et al.[30]， nanocarriers were used to introduce siRNAs into mammalian cells for the first time. Recently， Bao et al.[31] reported that positively charged delaminated layered double hydroxide lactate nanosheets （LDHlactateNS） exhibit a high adsorptive capacity for negatively charged biomolecules， including fluorescent dyes， forming neutral LDHnanosheet conjugates， and these neutral conjugates shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Nanoparticle mediated dsRNA delivery for gene silencing also has been successfully applied in mosquitos[32-33]， honey bees[34] and aphids[35].
Conclusions Exogenous dsRNAinduced gene silencing technique will become a research hotspot in pest control. The stability and persistence of dsRNA can be improved to some extent by the application of nanomaterials. Chemically modified molecules， nanoparticles， liposomes， and even viruses or bacteria can be used to deliver dsRNA， not only in plants but also in eukaryotic pathogens and pests.
However， the application of dsRNA to induce RNAi in practice production is inevitably limited by the costs， laws and regulations. Currently， exogenous dsRNA as a crop protection agent has not been commercialized. Commercial companies and researchers are looking to develop more efficient processes for largescale production of dsRNA， synthetically modified dsRNA or siRNA effector molecules[36]. Issues related to the specificity， entry， persistence and systematic movement of dsRNA in plants， offtarget effects and risk prediction remain to be fully addressed. In addition， policies and regulatory frameworks about the application of nanoparticlemediated delivery of dsRNA remain to be developed， to facilitate the commercialization of this material.
In summary， the application of exogenous dsRNA to induce plant resistance to viral disease is one of the effective methods to reduce the use of toxic pesticides in the environment， and to improve ecological sustainability and environment safety.
References
[1] KHALID A， ZHANG Q， YASIR M， et al. Small RNA based genetic engineering for plant viral resistance： application in crop protection[J]. Front Microbiol， 2017， 8： 43.
[2] IBRAHIM AB， ARAGAO FJ. RNAimediated resistance to viruses in genetically engineered plants[J]. Plant Gene Silencing： Methods and Protocols， 2015， 287： 81-92.
[3] DIETZGEN R， MITTER N. Transgenic gene silencing strategies for virus control[J]. Australas Plant Pathol， 2006， 35： 605-618.
[4] DUTTA TK， BANAKAR P， RAO U. The status of RNAibased transgenic research in plant nematology[J]. Front Microbiol， 2015， 5760.
[5] TAMILARASAN S， RAJAM M. Engineering crop plants for nematode resistance through hostderived RNA interference[J]. Cell Dev Biol， 2013， 2： 114.
[6] SCOTT JG， MICHEL K， BARTHOLOMAY LC， et al. Towards the elements of successful insect RNAi[J]. J Insect Physiol， 2013， 59： 1212-1221.
[7] YU N， CHRISTIAENS O， LIU J， et al. Delivery of dsRNA for RNAi in insects： an overview and future directions[J]. Insect Sci， 2012， 20： 4-14.
[8] DANG Y， YANG Q， XUE Z， et al. RNA interference in fungi： pathways， functions， and applications[J]. Eukaryot Cell， 2011， 10： 1148-1155. [9] DUAN CG， WANG CH， GUO HS. Application of RNA silencing to plant disease resistance[J]. Silence， 2012， 3： 5.
[10] BAULCOMBE D. RNA silencing in plants[J]. Nature， 2004， 431： 356-363.
[11] BORGES F， MARTIENSSEN RA. The expanding world of small RNAs in plants[J]. Nat Rev Mol Cell Biol， 2015， 16： 727-741.
[12] ROBINSON KE， WORRALL EA， MITTER N. Double stranded RNA expression and its topical application for nontransgenic resistance to plant viruses[J]. Plant Biochem Biotechnol， 2014， 23： 231-237.
[13] JIANG L， DING L， HE B， et al. Systemic gene silencing in plants triggered by fluorescent nanoparticledelivered doublestranded RNA[J]. Nanoscale， 2014， 6： 9965-9969.
[14] TENLLADO F， DIAZRUIZ JR. Doublestranded RNAmediated interference with plant virus infection[J]. Journal of Virology， 2001， 75： 12288-12297.
[15] TIMMONS L， FIRE A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene， 2001， 263： 103-112.
[16] TAKIFF HE， CHEN S. Genetic analysis of the rnc operon of Escherichia coli[J]. Bacteriol， 1989， 171： 2581-2590.
[17] VOLOUDAKIS AE， HOLEVA MC， SARIN LP， et al. Efficient doublestranded RNA production methods for utilization in plant virus control[J]. Plant Virology Protocols[J]. Springer; 2015： 255-274.
[18] TENLLADO F， MARTI‘NEZGARCI’A B， VARGAS M， et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections[J]. BMC Biotechnol， 2003， 3： 3.
[19] TENLLADO F， LLAVE C， DIAZRUIZ JR.RNA interference as a new biotechnological tool for the control of virus diseases in plants[J]. Virus Res， 2004， 102： 85-96.
[20] YIN G， SUN Z， LIU N， et al. Production of doublestranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system[J]. Appl Microbiol Biotechnol， 2009， 84： 323-333.
[21] GAN D， ZHANG J， JIANG H， et al. Bacterially expressed dsRNA protects maize against SCMV infection[J]. Plant Cell Rep， 2010， 29： 1261-1268.
[22] CARBONELL A， DE ALBA AEM， FLORES R， et al. Doublestranded RNA interferes in a sequencespecific manner with the infection of representative members of the two viroid families[J]. Virology， 2008， 371： 44-53.
[23] KONAKALLA NC， KALDIS A， BERBATI M， et al. Exogenous application of doublestranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco[J]. Planta， 2016， 244： 961-969. [24] MITTER N， WORRALL EA， ROBINSON KE， et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses[J]. Nat Plants， 2017， 3： 16207.
[25] XU ZP， STEVENSON GS， LU CQ， et al. Stable suspension of layered double hydroxide nanoparticles in aqueous solution[J]. Am Chem Soc， 2006， 128： 36-37.
[26] LAU SE， SCHWARZACHER T， OTHMAN RY， et al. dsRNA silencing of an R2R3MYB transcription factor affects flower cell shape in a Dendrobium hybrid[J]. BMC Plant Biol， 2015， 15： 194.
[27] KIM YD， PARK TE， SINGH B， et al. Nanoparticlemediated delivery of siRNA for effective lung cancer therapy[J]. Nanomedicine， 2015， 10： 1165-1188.
[28] YOUNG SW， STENZEL M， YANG JL. NanoparticlesiRNA： a potential cancer therapy[J]. Crit Rev Oncol Hematol， 2016， 98： 159-169.
[29] LADEWIG K， NIEBERT M， XU ZP， et al. Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles[J]. Biomaterials， 2010， 31： 1821-1829.
[30] WONG Y， MARKHAM K， XU ZP， et al. Efficient delivery of siRNA to cortical neurons using layered double hydroxide nanoparticles[J]. Biomaterials， 2010， 31： 8770-8779.
[31] BAO W， WANG J， WANG Q， et al. Layered double hydroxide nanotransporter for molecule delivery to intact plant cells[J]. Sci Rep， 2016， 6： 26738.
[32] ZHANG X， MYSORE K， FLANNERY E， et al. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae[J]. Journal of Visualized Experiments Jove， 2015， 97 （97）： e52523-e52523.
[33] DAS S， DEBNATH N， CUI Y， et al. Chitosan， carbon quantum dot， and silica nanoparticle mediated dsRNA delivery for gene silencing in Aedes aegypti： a comparative analysis[J]. ACS Appl Mater Interfaces， 2015， 7： 19530-19535.
[34] LIBYARLAY H， LI Y， STROUD H， et al. RNA interference knockdown of DNA methyltransferase 3 affects gene alternative splicing in the honey bee[J]. Proc Natl Acad Sci U S A， 2013， 110： 12750-12755.
[35] THAIRU M， SKIDMORE I， BANSAL R， et al. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species[J]. Insect Mol Biol， 2017， 26： 356-368.
[36] PALLI SR. RNA interference in Colorado potato beetle： steps toward development of dsRNA as a commercial insecticide[J]. Curr Opin Insect Sci， 2014， 6： 1-8.
Key words Plant; dsRNA; Induction; Virus; Resistance
With the development of biotechnology， an increasing research effort has been devoted to exploring the pest and disease resistance of crops in recent years. As a phenomenon inherent in living organisms， RNA silencing plays a decisive role in growth， development， and host defense against viral invasion. RNA silencing has been shown to be an effective way to protect plants against viruses， viroids， nematodes， pests and fungi[1-9]. Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. A target gene in a biological cell can be specifically silenced by inducing an exogenous homologous sequence， without influencing the expression of other genes. Gene silencing can occur during transcription （transcriptional gene silencing， TGS） or after transcription （posttranscriptional gene silencing， PTGS）[10-11].
Plant virus diseases can greatly reduce crop yield and affect local and global food security. The challenges faced by virus disease prevention and control include the lack of resistance genes， the emergence of new plant viruses， insecticide resistance of insects that spread viruses， and the implementation of disease control measures in farmland， as well as the lack of plant antiviral agents.
RNAmediated resistance to viruses is highly safe to the environment. No viral proteins can be synthesized in the cells of plants that have been transformed with untranslated genes or nucleotide sequences， which avoids the occurrence of heterologous encapsidation. These types of resistance， however， seem to be effective only against viral strains closely related to the source of the transgene. To endow the plants with resistance against multiple viral species， the effective nucleic acid sequences of several viruses are ligated together and transformed to plants. However， such method is expensive， time consuming， but also requires efficient protocols for plant transformation. In addition， genetically modified plants， especially genetically modified food crops have not been well accepted by the public. In order to overcome these limitations and public concerns about genetically modified food crops， dsRNA is being explored as a topical application to trigger the RNAi pathway against pathogenic viruses[12]. Viral RNA Silencing Induced by Exogenous Doublestranded RNA
Recent research reports have shown that RNAimediated viral silencing in plants can be achieved by introducing exogenous dsRNA[13-14]. Tenllado and DiazRuiz[14] reported that dsRNA derived from viral sequences could interfere with virus infection in a sequencespecific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacteriummediated transientexpression assay. For example， mechanical inoculation of pepper mild mottle virus （PMMoV） and dsRNA homologous to PMMoV replicase prevented the formation of virusinduced lesions on the inoculated leaves of Nicotiana tabacum cv. Xanthi or Capsicum chinense.
Tenllado and DiazRuiz[14] also reported that dsRNAinduced viral immunity is dependent on the sequence specificity and minimum homology length between the dsRNA and the target virus. For example， a 0.6-1.0 kb dsRNA fragment that is 100% homologous to PMMoV is capable of inhibiting viral infection， and a dsRNA fragment less than 300 bp in length or low in homology shows a reduced ability to inhibit viral infection.
The dsRNA fragments used to induce resistance against viruses were artificially synthetic in most initial studies. Then， it was found that a large amount of dsRNA can be produced using an RNase IIIdeficient strain HT115 of Escherichia coli[15-16]. Crude extracts of the bacterially expressed dsRNA can be used to protect plants against the infection of viruses such as PMMoV and PPV in tobaccos[17-18].
The application of dsRNA in crop disease control has drawn more and more attention since its first report[14， 18-20]. In the study of Gan et al.[21]， two fragments of the Sugarcane Mosaic Virus （SCMV） CP （coat protein） gene were cloned into the invertedrepeat cloning vector， and transformed individually into E. coli HT115， and then the crude extracts of E. coli HT115 containing large amounts of dsRNA were sprayed to plants， and the results demonstrated that spraying crude dsRNAcontaining extracts inhibited SCMV infection. In addition， this study also showed that spraying plants with SCMV CP1 dsRNA provided protective effect against viral infection for only five days， which was consistent with the findings of Tenllado[14].
However， the mechanisms of uptake and spread of dsRNA， and how they are converted into siRNAs are still poorly understood[22]. A recent study by Konakalla et al.[23] showed that exogenously applied dsRNA exhibits a rapid systemic trafficking in planta， and it is processed successfully by DICERlike proteins producing siRNAs. This study also showed the fast systemic spread of TMV p126 dsRNA from the treated （local） to nontreated （systemic） leaves beginning from 1 h postapplication， and stemloop RTPCR confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves postapplication. From all above studies， it can be concluded that systemic RNAi resistance against virus can be induced by sequencespecific dsRNA. However， the instability of dsRNA is still a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only five days.
Nanoparticlemediated Delivery of dsRNA
The instability of dsRNA sprayed onto plant leaves is still the biggest challenge in viral control using dsRNA. In the study of Mitter et al.[24]， a new layered double hydroxide （LDH） nanomaterial was used as a carrier for topical delivery of RNAi for sustained protection against plant viruses， which can not only improves the stability of dsRNA， but also result in slow release of dsRNA over several weeks.
LDH nanosheets which are nontoxic， biodegradable and biocompatible[25]， can load dsRNA to form dsRNALDH complexes referred to as BioClay[24]. After BioClay is sprayed onto plant leaves， it slowly degrades under the action of carbon dioxide and water in the atmosphere， and release dsRNA. The LDHcombined dsRNA cannot be easily eluted from plant leaves， and can provide protection against viruses for 30 days.
Previous studies[13， 24， 26] have shown that dsRNA can be taken up by plant cells and induce silencing of an endogenous gene. One explanation is that dsRNA is rapidly transported into other cells after being absorbed. Significantly， a single spray of dsRNA loaded on LDH （BioClay） afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves[24]. The mechanisms of action on the absorption， transport and processing of dsRNA are still poorly understood. Nanoparticlemediated RNAi in cancer prevention and treatment has become a research hotspot in recent years[27-28].
In the studies of Ladewig et al.[29] and Wong et al.[30]， nanocarriers were used to introduce siRNAs into mammalian cells for the first time. Recently， Bao et al.[31] reported that positively charged delaminated layered double hydroxide lactate nanosheets （LDHlactateNS） exhibit a high adsorptive capacity for negatively charged biomolecules， including fluorescent dyes， forming neutral LDHnanosheet conjugates， and these neutral conjugates shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Nanoparticle mediated dsRNA delivery for gene silencing also has been successfully applied in mosquitos[32-33]， honey bees[34] and aphids[35].
Conclusions Exogenous dsRNAinduced gene silencing technique will become a research hotspot in pest control. The stability and persistence of dsRNA can be improved to some extent by the application of nanomaterials. Chemically modified molecules， nanoparticles， liposomes， and even viruses or bacteria can be used to deliver dsRNA， not only in plants but also in eukaryotic pathogens and pests.
However， the application of dsRNA to induce RNAi in practice production is inevitably limited by the costs， laws and regulations. Currently， exogenous dsRNA as a crop protection agent has not been commercialized. Commercial companies and researchers are looking to develop more efficient processes for largescale production of dsRNA， synthetically modified dsRNA or siRNA effector molecules[36]. Issues related to the specificity， entry， persistence and systematic movement of dsRNA in plants， offtarget effects and risk prediction remain to be fully addressed. In addition， policies and regulatory frameworks about the application of nanoparticlemediated delivery of dsRNA remain to be developed， to facilitate the commercialization of this material.
In summary， the application of exogenous dsRNA to induce plant resistance to viral disease is one of the effective methods to reduce the use of toxic pesticides in the environment， and to improve ecological sustainability and environment safety.
References
[1] KHALID A， ZHANG Q， YASIR M， et al. Small RNA based genetic engineering for plant viral resistance： application in crop protection[J]. Front Microbiol， 2017， 8： 43.
[2] IBRAHIM AB， ARAGAO FJ. RNAimediated resistance to viruses in genetically engineered plants[J]. Plant Gene Silencing： Methods and Protocols， 2015， 287： 81-92.
[3] DIETZGEN R， MITTER N. Transgenic gene silencing strategies for virus control[J]. Australas Plant Pathol， 2006， 35： 605-618.
[4] DUTTA TK， BANAKAR P， RAO U. The status of RNAibased transgenic research in plant nematology[J]. Front Microbiol， 2015， 5760.
[5] TAMILARASAN S， RAJAM M. Engineering crop plants for nematode resistance through hostderived RNA interference[J]. Cell Dev Biol， 2013， 2： 114.
[6] SCOTT JG， MICHEL K， BARTHOLOMAY LC， et al. Towards the elements of successful insect RNAi[J]. J Insect Physiol， 2013， 59： 1212-1221.
[7] YU N， CHRISTIAENS O， LIU J， et al. Delivery of dsRNA for RNAi in insects： an overview and future directions[J]. Insect Sci， 2012， 20： 4-14.
[8] DANG Y， YANG Q， XUE Z， et al. RNA interference in fungi： pathways， functions， and applications[J]. Eukaryot Cell， 2011， 10： 1148-1155. [9] DUAN CG， WANG CH， GUO HS. Application of RNA silencing to plant disease resistance[J]. Silence， 2012， 3： 5.
[10] BAULCOMBE D. RNA silencing in plants[J]. Nature， 2004， 431： 356-363.
[11] BORGES F， MARTIENSSEN RA. The expanding world of small RNAs in plants[J]. Nat Rev Mol Cell Biol， 2015， 16： 727-741.
[12] ROBINSON KE， WORRALL EA， MITTER N. Double stranded RNA expression and its topical application for nontransgenic resistance to plant viruses[J]. Plant Biochem Biotechnol， 2014， 23： 231-237.
[13] JIANG L， DING L， HE B， et al. Systemic gene silencing in plants triggered by fluorescent nanoparticledelivered doublestranded RNA[J]. Nanoscale， 2014， 6： 9965-9969.
[14] TENLLADO F， DIAZRUIZ JR. Doublestranded RNAmediated interference with plant virus infection[J]. Journal of Virology， 2001， 75： 12288-12297.
[15] TIMMONS L， FIRE A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene， 2001， 263： 103-112.
[16] TAKIFF HE， CHEN S. Genetic analysis of the rnc operon of Escherichia coli[J]. Bacteriol， 1989， 171： 2581-2590.
[17] VOLOUDAKIS AE， HOLEVA MC， SARIN LP， et al. Efficient doublestranded RNA production methods for utilization in plant virus control[J]. Plant Virology Protocols[J]. Springer; 2015： 255-274.
[18] TENLLADO F， MARTI‘NEZGARCI’A B， VARGAS M， et al. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections[J]. BMC Biotechnol， 2003， 3： 3.
[19] TENLLADO F， LLAVE C， DIAZRUIZ JR.RNA interference as a new biotechnological tool for the control of virus diseases in plants[J]. Virus Res， 2004， 102： 85-96.
[20] YIN G， SUN Z， LIU N， et al. Production of doublestranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system[J]. Appl Microbiol Biotechnol， 2009， 84： 323-333.
[21] GAN D， ZHANG J， JIANG H， et al. Bacterially expressed dsRNA protects maize against SCMV infection[J]. Plant Cell Rep， 2010， 29： 1261-1268.
[22] CARBONELL A， DE ALBA AEM， FLORES R， et al. Doublestranded RNA interferes in a sequencespecific manner with the infection of representative members of the two viroid families[J]. Virology， 2008， 371： 44-53.
[23] KONAKALLA NC， KALDIS A， BERBATI M， et al. Exogenous application of doublestranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco[J]. Planta， 2016， 244： 961-969. [24] MITTER N， WORRALL EA， ROBINSON KE， et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses[J]. Nat Plants， 2017， 3： 16207.
[25] XU ZP， STEVENSON GS， LU CQ， et al. Stable suspension of layered double hydroxide nanoparticles in aqueous solution[J]. Am Chem Soc， 2006， 128： 36-37.
[26] LAU SE， SCHWARZACHER T， OTHMAN RY， et al. dsRNA silencing of an R2R3MYB transcription factor affects flower cell shape in a Dendrobium hybrid[J]. BMC Plant Biol， 2015， 15： 194.
[27] KIM YD， PARK TE， SINGH B， et al. Nanoparticlemediated delivery of siRNA for effective lung cancer therapy[J]. Nanomedicine， 2015， 10： 1165-1188.
[28] YOUNG SW， STENZEL M， YANG JL. NanoparticlesiRNA： a potential cancer therapy[J]. Crit Rev Oncol Hematol， 2016， 98： 159-169.
[29] LADEWIG K， NIEBERT M， XU ZP， et al. Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles[J]. Biomaterials， 2010， 31： 1821-1829.
[30] WONG Y， MARKHAM K， XU ZP， et al. Efficient delivery of siRNA to cortical neurons using layered double hydroxide nanoparticles[J]. Biomaterials， 2010， 31： 8770-8779.
[31] BAO W， WANG J， WANG Q， et al. Layered double hydroxide nanotransporter for molecule delivery to intact plant cells[J]. Sci Rep， 2016， 6： 26738.
[32] ZHANG X， MYSORE K， FLANNERY E， et al. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae[J]. Journal of Visualized Experiments Jove， 2015， 97 （97）： e52523-e52523.
[33] DAS S， DEBNATH N， CUI Y， et al. Chitosan， carbon quantum dot， and silica nanoparticle mediated dsRNA delivery for gene silencing in Aedes aegypti： a comparative analysis[J]. ACS Appl Mater Interfaces， 2015， 7： 19530-19535.
[34] LIBYARLAY H， LI Y， STROUD H， et al. RNA interference knockdown of DNA methyltransferase 3 affects gene alternative splicing in the honey bee[J]. Proc Natl Acad Sci U S A， 2013， 110： 12750-12755.
[35] THAIRU M， SKIDMORE I， BANSAL R， et al. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species[J]. Insect Mol Biol， 2017， 26： 356-368.
[36] PALLI SR. RNA interference in Colorado potato beetle： steps toward development of dsRNA as a commercial insecticide[J]. Curr Opin Insect Sci， 2014， 6： 1-8.