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RNAi technology for plant protection and its application in wheat

RNAi technology for plant protection and its application in wheat aBIOTECH (2021) 2:365–374 https://doi.org/10.1007/s42994-021-00036-3 aBIOTECH REVIEW RNAi technology for plant protection and its application in wheat 1 1 1 2 1,3& Shaoshuai Liu , Shuaifeng Geng , Aili Li , Yingbo Mao , Long Mao Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Chinese Academy of Sciences (CAS) Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, University of CAS, Chinese Academy of Sciences, Shanghai 200032, China Sino-Agro Research Station for Salt Tolerant Crops, Yellow River Delta, Kenli District, Dongying 257500, Shandong, China Received: 29 December 2020 / Accepted: 23 February 2021 / Published online: 11 March 2021 Abstract The RNAi technology takes advantage of the intrinsic RNA interference (RNAi) mechanism that exists in nearly all eukaryotes in which target mRNAs are degraded or functionally suppressed. Significant progress has been made in recent years where RNAi technology is applied to several crops and economic plants for protection against diseases like fungi, pests, and nematode. RNAi technology is also applied in controlling pathogen damages in wheat, one of the most important crops in the world. In this review, we first give a brief introduction of the RNAi technology and the underneath mechanism. We then review the recent progress of its utilization in crops, particular wheat. Finally, we discuss the existing challenges and prospect future development of this technology in crop protection. Keywords Double-stranded RNA, Pathogens, Pests, Nematodes, RNA interference, Small RNA, Wheat INTRODUCTION scope of Bt crops and the appearance of Bt-resistant pests in fields call for new technologies for pest control Wheat (Triticum aestivum L.) contributes more than (Carriere et al. 2015; Jin et al. 2015; Tabashnik et al. 20% of the total dietary calories and proteins for 2013). The phenomenon of RNA interference (RNAi) is humans worldwide (Shiferaw et al. 2013). It plays a widely found in eukaryotes (plants, fungi, insects, ani- pivotal role in securing the global food demand. The mals, and nematodes etc.) and has been developed as a increase of wheat yield, however, has slowed down in promising technology for crop health protection (Zhang recent years partly due to newly emerging varieties of et al. 2017). RNAi is a natural process that involves the various diseases—pathogens, pests and nematodes regulation of gene expression by several manners: (Rosegrant and Cline 2003). On the other hand, the effective post-transcriptional gene silencing (PTGS), overuse of pesticides for disease control has posed translational inhibition, RNA destabilization, and/or substantial risks to food safety, the environment, and all transcriptional gene silencing (TGS) by directing DNA living organisms (Ali 2014). The transgenic crops methylation (Fire et al. 1998; Coleman et al. 2015; expressing insecticidal proteins from Bacillus Ghildiyal et al. 2008; Huvenne and Smagghe 2010; thuringiensis (Bt) effectively reduced the insecticide Jones-Rhoades et al. 2006; Liu et al. 2020; Mao et al. usage and increased crop yields. However, the limited 2007; Sherman et al. 2015). Here, we review recent progress in the development of RNAi-based plant pro- tection technologies, particularly on its application in & Correspondence: maolong@caas.cn (L. Mao) The Author(s) 2021 366 aBIOTECH (2021) 2:365–374 wheat. We discuss its potential for the control of fungal in Caenorhabditis elegans (Sijen et al. 2001) and fungi pathogens, pests and nematodes, as well as current (Dang et al. 2011); however, a similar RdRP-based challenges facing RNAi strategy. We also prospect the amplification system is yet to be discovered in insects future improvement in delivery methods for effective (Zotti et al. 2018). Given the presence of RNAi pathways applications of this technology in crop protection. in pathogens, pests, and nematodes, it is not surprising to take advantage of its working mechanism in crop protection. THE MECHANISM OF RNAI TECHNOLOGY RNAi is a self-protection mechanism in eukaryotic cells DELIVERY OF INTERFERING RNAS and is triggered by double-stranded RNA (dsRNA) when present in a cell. dsRNA is processed by the ribonucle- Interspecific transportation of sRNAs takes place natu- ase III enzyme Dicer or Dicer-like enzymes to produce rally. siRNAs can be shuttled between plants and small interfering RNAs (siRNAs) of 20–30 nucleotide pathogens by secreted vesicles (Cai et al. 2018; Weiberg (nt) long. These small RNA (sRNA) are bound to Arg- et al. 2013). In cotton, the production of microRNAs onaute family proteins (AGOs), the catalytic components (miRNAs) miR166 and miR159 was increased upon of the RNAi system. The AGO/siRNA complexes are then Verticillium dahliae (a vascular fungal pathogen recruited to the RNA-induced silencing complex (RISC) responsible for devastating wilt diseases in many crops) (Lee et al. 2010), which mediates mRNA degradation, infection and transported to infection sites to silence mRNA translation, or chromatin modification (Borges virulence genes reducing its damage (Zhang et al. 2016). and Martienssen 2015) (Fig. 1). In most eukaryotes, Despite these in vivo mechanisms, the RNAi technology including pathogens and pests, RNA-dependent RNA is impeded by in vitro dsRNA delivering efficiency. polymerases (RdRPs) have been identified for sec- Numerous efforts on artificial delivery methods have ondary dsRNA synthesis and are essential for the sys- been attempted. The selection of the suitable delivery temic effect of RNAi. Two works have specified approaches (e.g. host-induced gene silencing, foliar functions for the RdRP activity in RNAi sprays, recombinant microbes) is in fact determining Fig. 1 The RNAi technology and its application in crop diseases control. (Left) Crop disease control by RNAi. dsRNA delivery strategies for wheat protection mainly via HIGS, foliar sprays and recombinant microbes. Each of these strategies contains advantages, relying on the specific condition involved. Additional methods are also used such as nanoparticles, baits, trunk injection, and root soaking. (Right) The RNAi mechanism. Double-stranded RNA (dsRNA) molecule binds to a Dicer/Dcl protein, which cleaves it into small interfering RNAs (siRNAs); these siRNAs bind to an Argonaute (AGO) protein, to form an RNA-Induced Silencing Complex (RISC), then RISC separates the siRNAs into two strands. The siRNA/RISC complex binds the complementary sequence of the target mRNA resulting in post- transcriptional gene silencing (PTGS) via degradation of the mRNA target (indicated by a scissor) or inhibition of its translation (indicated by red vertical bar), or resulting in transcriptional gene silencing (TGS) in the nucleus via chromatin modifications. In fungi and nematodes, the silencing signal can be perpetuated by the action of the RNA-dependent RNA polymerase (RdRP) for siRNA secondary amplification, but for pests, RdRP is not yet found The Author(s) 2021 aBIOTECH (2021) 2:365–374 367 the success of the technology (Fig. 1). A few methods persistent effects were obtained for several common have been tested. species (Baum et al. 2007; Mao and Zeng 2014; Sun The first approach is the application of synthetic et al. 2019; Zhu et al. 2012). The phloem-feeding dsRNA or sRNA derived from pathogen or pest genes as hemipterans such as aphids with specialized mouth- pesticides on crop leaves. Foliar application with parts (stylets) that penetrate through plant tissues to sprayable RNAi-based products, such as sRNAs, is suit- ingest cell saps. In this case, dsRNA sequences of shp able for controlling pests and pathogens on stems, gene effectively reduced the growth, the reproduction, foliage, or fruits. The products are evaluated similarly to and the survival rate of tested aphids. Remarkably, other topical pesticides where the RNA solution is sprayed on developmental aberrations were also observed such as leaves, or fed to the target pests, and impacts on insects winged adults and delayed maturation (Abdellatef et al. are then observed (Andrade and Hunter 2017). One of 2015). This method is a complementary tool to Bt-based the first case exploring the applications of sprayable insect-resistant plants which is not effective for several RNA molecules to control pests was the use of siRNA hemipterans with specialized stylets. Cotton plants against the diamondback moth (Plutella xylostella). constitutively expressing dsRNA from genes encoding Brassica leaves that were sprayed with chemically syn- the P450 protein CYP6AE14 and NDPH dehydrogenase thesized siRNAs targeting the acetylcholine esterase protein 2 of cotton bollworm (Helicoverpa armigera) gene AchE2 caused high mortality for P. xylostella. (Gong significantly improved resistance to this pest, and the et al. 2013). In another case, foliar application of dsRNA dsNDPH cotton is almost equivalent to Bt cottons in targeting the cytochrome P450 (CYP3) gene of Fusarium resistance efficiency (Mao et al. 2011;Wu etal. 2016). graminearum resulted in successful inhibition of fungal Similarly, dsRNA homologous to V-type ATPase gene of growth in directly sprayed leaves as well as the distal corn root worm (Diabrotica virgifera) in transgenic corn non-sprayed leaves in barley plants (Koch et al. 2016). plants rendered significant improvement of insect This strategy or so-called spray-induced gene silencing resistance (Baum et al. 2007). (SIGS) opens an avenue of development of biopesticide For woody plants, such as fruit trees, dsRNA can be which is environmentally friendly. Moreover, since RNAi delivered via insecticidal baits, nanoparticle trunk is highly dependent on the sequence specificity, it has injection and root soaking. The information of these little effects on the non-target microorganisms or non- methods can be found elsewhere for detail (Liu et al. target pests. 2020; Zhu and Palli 2019). The second method is to use recombinant microbes such as virus and bacteria engineered to produced dsRNA in host crops (Cagliari et al. 2018; Dubrovina and THE APPLICATION OF RNAI FOR WHEAT Kiselev 2019; Goulin et al. 2019). Virus-induced gene PROTECTION silencing (VIGS) is a naturally occurring (Baulcombe 2015; Waterhouse et al. 2001). Unlike stable RNAi and Management of bacterial and fungal pathogens mutants, the transiently expressed dsRNA by VIGS does not modify plant genetic composition. For instance, In wheat, a few serious wheat diseases, such as Fusar- three midgut-expressed CYP genes of the Lepidoptera ium head blight (FHB) caused by necrotrophic fungi of insect, Manduca sexta were targeted through viral vec- the genus Fusarium and leaf rust caused by biotrophic tors to produce dsRNA in the host plant. The viral vector fungi of the genus Puccinia (Table 1), have been targeted was engineered using Tobacco Rattle Virus (TRV) to using RNAi technology. Transgenic wheat plants were deliver dsRNA into Nicotiana attenuata (Kumar et al. engineered to confer three hairpin RNA fragments 2012). DsRNA could also be produced in the bacteria derived from the Fusarium graminearum chitin synthase (HT115). When the cotton bollworm (Helicoverpa gene (Chs3b), which is responsible for the biosynthesis armigera) larvae fed with the artificial diet coated with of chitin. These transgenic plants showed strong resis- dsRNA expressing HT115, high mortality was observed tance to FHB and Fusarium seedling blight (FSB) (Cheng after five days. Data showed that inhibition of target et al. 2015). On the other hand, expressing dsRNA gene expression led to significant reductions in body complementary to mRNAs of Puccinia triticina MAP-ki- weight, body length, and pupation rate (Ai et al. 2018). nase (PtMAPK1, 520 bp) or a cyclophilin (PtCYC1, The third approach is host-induced gene silencing 501 bp) showed efficient silencing of the corresponding (HIGS) which employs transgenic plants to produce genes in the fungus and significant reduction of the dsRNA derived from pathogen or pest genes. RNAi fungal pathogenicity and growth in transgenic wheat. P. occurs in pests when they ingest sufficient dsRNA or triticina is an aggressive fungal pathogen that causes sRNA. Tests have been made for a few pests where severe leaf rust disease in wheat. P. triticina The Author(s) 2021 368 aBIOTECH (2021) 2:365–374 Table 1 RNAi target genes tested in pests/pathogens/nematodes Organism Target genes Assay/ Effects References method Insects Sitobion avenae Salivary sheath protein (SHP) HIGS Mortality/ Abdellatef et al. fecundity/transgenetional (2015) gene silencing Rhopalosiphum Acetylcholinesterase gene RpAce1 Injection Susceptibility/fecundity Xiao et al. padi (2015) Sitobion avenae Catalase gene CAT Feeding Mortality Deng and Zhao (2014) Sitobion avenae Acetylcholinesterase gene SaAce1 Injection Susceptibility/fecundity Xiao et al. (2015) Sitobion avenae Cytochrome c oxidase subunit VIIc precursor, Feeding Mortality/developmental Zhang et al. zinc finger protein, three unknown proteins stunting (2013) Sitobion avenae Secreted salivary peptide DSR32, salivary Feeding Mortality Wang et al. protein DSR33, serine protease 1 DSR48 (2015) Sitobion avenae Olfactory coreceptor gene SaveOrco Feeding Impaired response Fan et al. (2015) Sitobion avenae Lipase maturation factor 2-like gene HIGS Mortality/fecundity Xu et al. (2017) Sitobion avenae Laccase 1 (Lac1) Feeding Mortality Zhang et al. (2018) Sitobion avenae Zinc finger protein (SaZFP) HIGS Mortality/transgenetional gene Sun et al. silencing (2019) Sitobion avenae Ecdysone receptor (EcR) and ultraspiracle Feeding Mortality/fecundity Yan et al. protein (USP) (2016) Sitobion avenae Chitin synthase 1 (CHS1) HIGS Mortality/fecundity Zhao et al. (2018) Pathogens Fusarium Cytochrome P450 lanosterol HIGS Inhibiting fungal mycelium Koch et al. graminearum formation (2013) C-14a-demethylase (CYP51) Fusarium Cytochrome P450 lanosterol C-14a- SIGS Inhibition of fungal growth Koch et al. graminearum demethylase CYP51 (2016) Fusarium Chs3b HIGS Restriction of fungal growth Cheng et al. graminearum through (2015) Blumeria graminis Virulence effector (Avra10) HIGS Reduced fungal development Nowara et al. (2010) Fusarium asiaticum Myosin 5 SIGS Reduced virulence Song et al. (2018) B. graminis f. sp. Ribonuclease-like protein HIGS Reduced virulence Pliego et al. hordei (2013) Ribonuclease-like protein Virulence effector Glucanase Metalloprotease Virulence effector Fusarium culmorum Secreted lipase (Fgl1), Mitogen-activated VIGS and Reduced virulence Chen et al. protein (MAP) kinase (Fmk1), Beta 1,3- HIGS (2016) Glucan synthase (Gls1) Puccinia striiformis Calcineurin homologue (PsCNA1, PsCNB1) VIGS Slower extension of fungal Yin et al. f. sp. tritici hyphae (2010) Puccinia striiformis Mitogen-activated protein kinase (MAPK1), VIGS Reduced virulence Panwar et al. f. sp. tritici Cyclophilin (CYC1), Calcineurin regulatory (2013) subunit (CNB) The Author(s) 2021 aBIOTECH (2021) 2:365–374 369 Table 1 continued Organism Target genes Assay/ Effects References method Puccinia striiformis Protein kinase A catalytic subunit (PsCPK1) VIGS Reduced virulence Qi et al. (2018) f. sp. tritici 3a1/f1 Blumeria Three virulence effectors (SvrPm ) HIGS Reduced virulence Schaefer et al. graminis f. (2020) sp. tritici Puccinia striiformis Glycine-serine-rich effector (PstGSRE1) HIGS Reduced virulence and Qi et al. f. sp. tritici increased H O accumulation (2019a) 2 2 Nematodes Meloidogyne Heat-shock protein 90, isocitrate lyase, HIGS Reduced reproduction Lilley et al. incognita Mi-cpl-1 (2007) Pratylenchus spp. Troponin C (pat-10) Soaking Reduced reproduction Tan et al. solution (2013) Calponin (unc-87) proliferation was significantly reduced together with confer durable broad-spectrum powdery mildew resis- decreasing fungal target gene transcript abundance and tance (Acevedo-Garcia et al. 2017). reduced biomass accumulation in RNAi-based resistant Wheat streak mosaic virus (WSMV) is another per- plants (Panwar et al. 2018). sistent threat to wheat production. Transgenic wheat Powdery mildew caused by Blumeria graminis f. sp. plants constitutively expressing a polycistronic cassette hordei in barley and B. graminis f. sp. tritici in wheat is a of five miR395 arms, known as FanGuard (FGmiR395), serious disease as well. Transgenic barley expressing were exploited to target five distinct regions of the virus dsRNA targeting the avirulence gene Avra10, which genome. The consequent transgenic plants showed corresponds to the resistance gene Mla10, showed nearly complete immunity to WSMV (Fahim et al. 2012). reduced fungal gene transcripts and severely affected In the other case, a segment of 272 bp sequence derived fungal development (Nowara et al. 2010). Silencing of from the coat protein of Triticum mosaic virus (TriMV) 1,3-b-glucanosyltransferase genes (BgGTF1 and was cloned into the hairpin expression vector and BgGTF2) via VIGS that was built on the barley stripe constitutively expressed in wheat. The engineered mosaic virus (BSMV) significantly slowed down the wheat plants showed stable resistance to TriMV (Fahim growth of the powdery mildew fungus (Qi et al. 2019b). et al. 2010). Mildew resistance locus o (Mlo) encodes a transmem- brane protein (Panstruga et al. 2005) that acts as a Management of wheat pests negative regulator to suppress plant immunity in uninfected tissues. It is also involved in protection Several major pests, such as grain aphid (Sitobion avenae), against cell death as well as in responses to biotic and bird cherry-oat aphid (Rhopalosiphum padi), and wheat abiotic stresses (Piffanelli et al. 2002). Down-regulation aphid (Schizaphis graminum), can cause severe yield loss of the TaMlo gene via VIGS resulted in the broad-spec- (Table 1)(Peairs 2008; Smith and Chuang 2014;Yuetal. trum powdery mildew resistance in wheat (Va´rallyay 2014). Transgenic wheat plants expressing a 198 bp et al. 2012). Recently, gene-editing technologies were fragment of dsRNA complementary to the zinc finger used to achieve similar effects. For instance, simulta- protein (SaZFP) of grain aphid can effectively increase its neous knockout of the three TaMlo homoeologues by mortality and reduce its daily fecundity (Sun et al. 2019). TALEN (transcription activator-like effector nuclease) In barley, dsRNA targeting the grain aphid gene encoding produced transgenic wheat plants that were highly salivary sheath protein (SHP), a pivotal component of the resistant to powdery mildew infection, another work stylet penetration process, effectively reduces the repro- produced transgenic wheat plants that carry mutations duction and survival rates of the aphid and the effect can in the TaMLO-A1 allele using the CRISPR-Cas9 technol- be transmitted for seven generations (Abdellatef et al. ogy (Wang et al. 2014). On the other hand, non-trans- 2015). Effects of additional target genes were also con- genic TILLING (Targeting Induced Lesions IN Genomes) firmed by feeding or direct injection into grain aphid, such plants with partial loss-of-function alleles of TaMlo as those encoding catalase, acetylcholinesterase1, The Author(s) 2021 370 aBIOTECH (2021) 2:365–374 cytochrome c oxidase subunit VIIc precursor, and zinc and within 7 days after their addition to aquatic sys- finger protein, and abnormally high mortality and devel- tems containing natural water and various types of opmental stunting were observed (Wang et al. 2015; sediment (Albright et al. 2017; Fischer et al. 2017). Zhang et al. 2013). Despite this, actin-dsRNA derived Colorado potato bee- tle (CPB) remained active for at least four weeks after Management of nematodes in wheat application to potato leaves. It suppressed CPB larval weight gain, delayed its development, and increased its Wheat parasites cause enormous yield losses and mortality (San Miguel and Scott 2016). Therefore, dis- threaten the quality of grains, including Heterodera secting the process of dsRNA degradation is helpful in avenae, H. filipjevi and H. latipons (Table 1) (Toumi and evaluating the potential effect of dsRNA in various Waeyenberge 2013). Targeting of the Ha18764 effector environments and target organisms. protein family genes of H. avenae by the VIGS-based RNAi approach significantly attenuated the parasitism Cost-effective methods for dsRNA production and reproduction status of H. avenae in wheat (Yang et al. 2019). Down-regulation by RNAi of pat-10 and For RNAi application to be practical for field use, the unc-87 genes on Thorne’s meadow nematode (Praty- major hurdle is to produce sufficient amount of dsRNA. lenchus thornei), which infects wheat roots, significantly The traditional dsRNA production method in the labo- reduced the reproduction of the worms (Tan et al. ratory is expensive and produces only a limited amount 2013). Moreover, RNAi in wheat can be stimulated by of dsRNA and thus is not practical for large-scale poly-component biostimulants derived from metabo- application needs (Ahn et al. 2019). Producing dsRNA in lites of various soil streptomycetes which up-regulate bacterial cells with RNaseIII deficiency seems to be an siRNAs and miRNAs in wheat plants. These small RNAs alternative. However, only a handful works have are complementary to cereal cyst nematode mRNA and demonstrated microbial-based dsRNA production. One hence suppress their reproduction providing resistance approach uses L1440-HT115 (DE3) system that has to wheat plants (Blyuss et al. 2019). been successfully applied in the RNAi of Mythimna separate (Das et al. 2015; Parsons et al. 2018; Zhang et al. 2010). With more research underway, the pro- CHALLENGES FOR USING RNAI TECHNOLOGY duction efficiency of this system should be augmented to meet market demands. While the outlook of using RNAi for plant protection appears to be promising, several issues need to be Off-target effects resolved before efficient practical applications. RNAi is a sequence homology-dependent mechanism. The stability of dsRNA Several studies show that siRNA is not always specific and can have off-target effects and thus is problematic One of the primary concerns for the use of RNA as a in disease management (Mamta and Rajam 2017). Some biopesticide is their stability, especially for the spray- target genes are highly conserved between species able dsRNA and siRNA applications. Microorganisms in which increases the likelihood of off-targets among the environment can degrade dsRNA prior to their them. The sequences of vATPaseA and vATPaseEfrom L. uptake by pathogens or pests. Rapid degradation of decemlineata, for instance, shared 83% and 79% dsRNA may occur by nucleases in the saliva, gut lumen, nucleotide-sequence identities to their counterparts in and/or haemolymph of pests as well (Allen and Walker Western Corn Rootworm (WCR), respectively. dsRNAs III 2012; Chung et al. 2018; CoGuan et al. 2018; Katoch from WCR vATPaseA and vATPaseE could reduce the and Thakur 2012; Kennedy et al. 2004; Luo et al. 2013). fitness of Colorado potato beetle (CPB; Leptinotarsa The high or low pH found in the gut lumens of some decemlineata) in a bioassay (Baum et al. 2007). Com- pests can also reduce dsRNA stability either directly or putational design program is needed for specific and indirectly by affecting the activity of gut nucleases systemic evaluation of non-target and off-target effects (Cooper et al. 2019). which should be further verified by additional bioas- Other environmental factors may exert different says. In addition, feeding studies revealed that dsRNAs effects on the stability of dsRNA and sRNA. Several of at least 60 nucleotide (nt) in length are necessary for works show that dsRNA is degraded to unde- an efficient RNAi response in D. virgifera (Bolognesi tectable levels within 48 h after their application on et al. 2012) and Tribolium castaneum (Wang et al. three types of soil (silt loam, loamy sand, and clay loam) 2019). A minimum of 21 nt was required for the size of The Author(s) 2021 aBIOTECH (2021) 2:365–374 371 siRNA for efficient protection against WCR and active lowering costs of the technology. There is no doubt that orthologs (Bachman et al. 2013). a new era of disease control based on RNAi technology for crop protection is right at the corner. RNAi resistance Acknowledgements We are grateful for the financial support by the National Natural Science Foundation of China (No. 31701429), Pests and pathogens can develop resistance to RNAi- National Key Program for Transgenic Research (No. based products through various mechanisms as they do 2016ZX08009-001), and the ‘Yellow Delta Scholarship’ from the for conventional biopesticides. Compared to conven- municipal government of Dongying. tional commercialized transgenic crops expressing Bt Author contributions LM and SSL conceived the topic. S.S.L toxins for pest management (James, 2010). RNAi-based drafted the first manuscript. SFG and ALL read the draft and gave strategy induces down-regulation of the target gene by substantial suggestions for revision, LM revised the manuscript in-complete resistance in most of cases. This may with the help from YBM reduce the selection pressure on the pathogen that may Compliance with ethical standards contribute to durable resistance. But genetic variation in pathogenic organisms may also cause single nucleotide Conflict of interest There are no conflicts of interest. polymorphisms (SNPs) in the target gene. The efficiency of RNAi would be cut down owing to the reduction of Ethical approval We declare that all materials and methods comply with required ethical standards. complementarity between the target gene and the dsRNA. Synonymous SNPs lead to nearly no fitness cost Open Access This article is licensed under a Creative Commons on the pathogens and pests, but the difference between Attribution 4.0 International License, which permits use, sharing, dsRNA and the original gene sequences reduces their adaptation, distribution and reproduction in any medium or for- mat, as long as you give appropriate credit to the original complementarity, causing reduced RNAi effect or RNAi author(s) and the source, provide a link to the Creative Commons resistance (Scott et al. 2013; Yu et al. 2016). Thereby, licence, and indicate if changes were made. The images or other the potential of RNAi resistance should be taken into third party material in this article are included in the article’s consideration in application. Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will CONCLUSIONS AND FUTURE PROSPECTS need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. In the past few years, we have seen diverse applications of RNAi in crop protection methodologies against pests, pathogens, and nematodes. RNAi technology has emerged as a promising new strategy for wheat pro- References tection either. The wide use of HIGS on a commercial scale appears possible soon. The major obstacles for the Abdellatef E, Will T, Koch A, Imani J, Vilcinskas A, Kogel KH (2015) HIGS strategy will be resolved, by optimal target and Silencing the expression of the salivary sheath protein causes fragment selection methods, highly efficient transfor- transgenerational feeding suppression in the aphid Sitobion avenae. 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Pest Manag Sci 74:1239–1250. https://doi.org/10. reduces the survivability and reproductive capacity of the 1002/ps.4813 The Author(s) 2021 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png aBIOTECH Springer Journals

RNAi technology for plant protection and its application in wheat

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aBIOTECH (2021) 2:365–374 https://doi.org/10.1007/s42994-021-00036-3 aBIOTECH REVIEW RNAi technology for plant protection and its application in wheat 1 1 1 2 1,3& Shaoshuai Liu , Shuaifeng Geng , Aili Li , Yingbo Mao , Long Mao Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Chinese Academy of Sciences (CAS) Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, University of CAS, Chinese Academy of Sciences, Shanghai 200032, China Sino-Agro Research Station for Salt Tolerant Crops, Yellow River Delta, Kenli District, Dongying 257500, Shandong, China Received: 29 December 2020 / Accepted: 23 February 2021 / Published online: 11 March 2021 Abstract The RNAi technology takes advantage of the intrinsic RNA interference (RNAi) mechanism that exists in nearly all eukaryotes in which target mRNAs are degraded or functionally suppressed. Significant progress has been made in recent years where RNAi technology is applied to several crops and economic plants for protection against diseases like fungi, pests, and nematode. RNAi technology is also applied in controlling pathogen damages in wheat, one of the most important crops in the world. In this review, we first give a brief introduction of the RNAi technology and the underneath mechanism. We then review the recent progress of its utilization in crops, particular wheat. Finally, we discuss the existing challenges and prospect future development of this technology in crop protection. Keywords Double-stranded RNA, Pathogens, Pests, Nematodes, RNA interference, Small RNA, Wheat INTRODUCTION scope of Bt crops and the appearance of Bt-resistant pests in fields call for new technologies for pest control Wheat (Triticum aestivum L.) contributes more than (Carriere et al. 2015; Jin et al. 2015; Tabashnik et al. 20% of the total dietary calories and proteins for 2013). The phenomenon of RNA interference (RNAi) is humans worldwide (Shiferaw et al. 2013). It plays a widely found in eukaryotes (plants, fungi, insects, ani- pivotal role in securing the global food demand. The mals, and nematodes etc.) and has been developed as a increase of wheat yield, however, has slowed down in promising technology for crop health protection (Zhang recent years partly due to newly emerging varieties of et al. 2017). RNAi is a natural process that involves the various diseases—pathogens, pests and nematodes regulation of gene expression by several manners: (Rosegrant and Cline 2003). On the other hand, the effective post-transcriptional gene silencing (PTGS), overuse of pesticides for disease control has posed translational inhibition, RNA destabilization, and/or substantial risks to food safety, the environment, and all transcriptional gene silencing (TGS) by directing DNA living organisms (Ali 2014). The transgenic crops methylation (Fire et al. 1998; Coleman et al. 2015; expressing insecticidal proteins from Bacillus Ghildiyal et al. 2008; Huvenne and Smagghe 2010; thuringiensis (Bt) effectively reduced the insecticide Jones-Rhoades et al. 2006; Liu et al. 2020; Mao et al. usage and increased crop yields. However, the limited 2007; Sherman et al. 2015). Here, we review recent progress in the development of RNAi-based plant pro- tection technologies, particularly on its application in & Correspondence: maolong@caas.cn (L. Mao) The Author(s) 2021 366 aBIOTECH (2021) 2:365–374 wheat. We discuss its potential for the control of fungal in Caenorhabditis elegans (Sijen et al. 2001) and fungi pathogens, pests and nematodes, as well as current (Dang et al. 2011); however, a similar RdRP-based challenges facing RNAi strategy. We also prospect the amplification system is yet to be discovered in insects future improvement in delivery methods for effective (Zotti et al. 2018). Given the presence of RNAi pathways applications of this technology in crop protection. in pathogens, pests, and nematodes, it is not surprising to take advantage of its working mechanism in crop protection. THE MECHANISM OF RNAI TECHNOLOGY RNAi is a self-protection mechanism in eukaryotic cells DELIVERY OF INTERFERING RNAS and is triggered by double-stranded RNA (dsRNA) when present in a cell. dsRNA is processed by the ribonucle- Interspecific transportation of sRNAs takes place natu- ase III enzyme Dicer or Dicer-like enzymes to produce rally. siRNAs can be shuttled between plants and small interfering RNAs (siRNAs) of 20–30 nucleotide pathogens by secreted vesicles (Cai et al. 2018; Weiberg (nt) long. These small RNA (sRNA) are bound to Arg- et al. 2013). In cotton, the production of microRNAs onaute family proteins (AGOs), the catalytic components (miRNAs) miR166 and miR159 was increased upon of the RNAi system. The AGO/siRNA complexes are then Verticillium dahliae (a vascular fungal pathogen recruited to the RNA-induced silencing complex (RISC) responsible for devastating wilt diseases in many crops) (Lee et al. 2010), which mediates mRNA degradation, infection and transported to infection sites to silence mRNA translation, or chromatin modification (Borges virulence genes reducing its damage (Zhang et al. 2016). and Martienssen 2015) (Fig. 1). In most eukaryotes, Despite these in vivo mechanisms, the RNAi technology including pathogens and pests, RNA-dependent RNA is impeded by in vitro dsRNA delivering efficiency. polymerases (RdRPs) have been identified for sec- Numerous efforts on artificial delivery methods have ondary dsRNA synthesis and are essential for the sys- been attempted. The selection of the suitable delivery temic effect of RNAi. Two works have specified approaches (e.g. host-induced gene silencing, foliar functions for the RdRP activity in RNAi sprays, recombinant microbes) is in fact determining Fig. 1 The RNAi technology and its application in crop diseases control. (Left) Crop disease control by RNAi. dsRNA delivery strategies for wheat protection mainly via HIGS, foliar sprays and recombinant microbes. Each of these strategies contains advantages, relying on the specific condition involved. Additional methods are also used such as nanoparticles, baits, trunk injection, and root soaking. (Right) The RNAi mechanism. Double-stranded RNA (dsRNA) molecule binds to a Dicer/Dcl protein, which cleaves it into small interfering RNAs (siRNAs); these siRNAs bind to an Argonaute (AGO) protein, to form an RNA-Induced Silencing Complex (RISC), then RISC separates the siRNAs into two strands. The siRNA/RISC complex binds the complementary sequence of the target mRNA resulting in post- transcriptional gene silencing (PTGS) via degradation of the mRNA target (indicated by a scissor) or inhibition of its translation (indicated by red vertical bar), or resulting in transcriptional gene silencing (TGS) in the nucleus via chromatin modifications. In fungi and nematodes, the silencing signal can be perpetuated by the action of the RNA-dependent RNA polymerase (RdRP) for siRNA secondary amplification, but for pests, RdRP is not yet found The Author(s) 2021 aBIOTECH (2021) 2:365–374 367 the success of the technology (Fig. 1). A few methods persistent effects were obtained for several common have been tested. species (Baum et al. 2007; Mao and Zeng 2014; Sun The first approach is the application of synthetic et al. 2019; Zhu et al. 2012). The phloem-feeding dsRNA or sRNA derived from pathogen or pest genes as hemipterans such as aphids with specialized mouth- pesticides on crop leaves. Foliar application with parts (stylets) that penetrate through plant tissues to sprayable RNAi-based products, such as sRNAs, is suit- ingest cell saps. In this case, dsRNA sequences of shp able for controlling pests and pathogens on stems, gene effectively reduced the growth, the reproduction, foliage, or fruits. The products are evaluated similarly to and the survival rate of tested aphids. Remarkably, other topical pesticides where the RNA solution is sprayed on developmental aberrations were also observed such as leaves, or fed to the target pests, and impacts on insects winged adults and delayed maturation (Abdellatef et al. are then observed (Andrade and Hunter 2017). One of 2015). This method is a complementary tool to Bt-based the first case exploring the applications of sprayable insect-resistant plants which is not effective for several RNA molecules to control pests was the use of siRNA hemipterans with specialized stylets. Cotton plants against the diamondback moth (Plutella xylostella). constitutively expressing dsRNA from genes encoding Brassica leaves that were sprayed with chemically syn- the P450 protein CYP6AE14 and NDPH dehydrogenase thesized siRNAs targeting the acetylcholine esterase protein 2 of cotton bollworm (Helicoverpa armigera) gene AchE2 caused high mortality for P. xylostella. (Gong significantly improved resistance to this pest, and the et al. 2013). In another case, foliar application of dsRNA dsNDPH cotton is almost equivalent to Bt cottons in targeting the cytochrome P450 (CYP3) gene of Fusarium resistance efficiency (Mao et al. 2011;Wu etal. 2016). graminearum resulted in successful inhibition of fungal Similarly, dsRNA homologous to V-type ATPase gene of growth in directly sprayed leaves as well as the distal corn root worm (Diabrotica virgifera) in transgenic corn non-sprayed leaves in barley plants (Koch et al. 2016). plants rendered significant improvement of insect This strategy or so-called spray-induced gene silencing resistance (Baum et al. 2007). (SIGS) opens an avenue of development of biopesticide For woody plants, such as fruit trees, dsRNA can be which is environmentally friendly. Moreover, since RNAi delivered via insecticidal baits, nanoparticle trunk is highly dependent on the sequence specificity, it has injection and root soaking. The information of these little effects on the non-target microorganisms or non- methods can be found elsewhere for detail (Liu et al. target pests. 2020; Zhu and Palli 2019). The second method is to use recombinant microbes such as virus and bacteria engineered to produced dsRNA in host crops (Cagliari et al. 2018; Dubrovina and THE APPLICATION OF RNAI FOR WHEAT Kiselev 2019; Goulin et al. 2019). Virus-induced gene PROTECTION silencing (VIGS) is a naturally occurring (Baulcombe 2015; Waterhouse et al. 2001). Unlike stable RNAi and Management of bacterial and fungal pathogens mutants, the transiently expressed dsRNA by VIGS does not modify plant genetic composition. For instance, In wheat, a few serious wheat diseases, such as Fusar- three midgut-expressed CYP genes of the Lepidoptera ium head blight (FHB) caused by necrotrophic fungi of insect, Manduca sexta were targeted through viral vec- the genus Fusarium and leaf rust caused by biotrophic tors to produce dsRNA in the host plant. The viral vector fungi of the genus Puccinia (Table 1), have been targeted was engineered using Tobacco Rattle Virus (TRV) to using RNAi technology. Transgenic wheat plants were deliver dsRNA into Nicotiana attenuata (Kumar et al. engineered to confer three hairpin RNA fragments 2012). DsRNA could also be produced in the bacteria derived from the Fusarium graminearum chitin synthase (HT115). When the cotton bollworm (Helicoverpa gene (Chs3b), which is responsible for the biosynthesis armigera) larvae fed with the artificial diet coated with of chitin. These transgenic plants showed strong resis- dsRNA expressing HT115, high mortality was observed tance to FHB and Fusarium seedling blight (FSB) (Cheng after five days. Data showed that inhibition of target et al. 2015). On the other hand, expressing dsRNA gene expression led to significant reductions in body complementary to mRNAs of Puccinia triticina MAP-ki- weight, body length, and pupation rate (Ai et al. 2018). nase (PtMAPK1, 520 bp) or a cyclophilin (PtCYC1, The third approach is host-induced gene silencing 501 bp) showed efficient silencing of the corresponding (HIGS) which employs transgenic plants to produce genes in the fungus and significant reduction of the dsRNA derived from pathogen or pest genes. RNAi fungal pathogenicity and growth in transgenic wheat. P. occurs in pests when they ingest sufficient dsRNA or triticina is an aggressive fungal pathogen that causes sRNA. Tests have been made for a few pests where severe leaf rust disease in wheat. P. triticina The Author(s) 2021 368 aBIOTECH (2021) 2:365–374 Table 1 RNAi target genes tested in pests/pathogens/nematodes Organism Target genes Assay/ Effects References method Insects Sitobion avenae Salivary sheath protein (SHP) HIGS Mortality/ Abdellatef et al. fecundity/transgenetional (2015) gene silencing Rhopalosiphum Acetylcholinesterase gene RpAce1 Injection Susceptibility/fecundity Xiao et al. padi (2015) Sitobion avenae Catalase gene CAT Feeding Mortality Deng and Zhao (2014) Sitobion avenae Acetylcholinesterase gene SaAce1 Injection Susceptibility/fecundity Xiao et al. (2015) Sitobion avenae Cytochrome c oxidase subunit VIIc precursor, Feeding Mortality/developmental Zhang et al. zinc finger protein, three unknown proteins stunting (2013) Sitobion avenae Secreted salivary peptide DSR32, salivary Feeding Mortality Wang et al. protein DSR33, serine protease 1 DSR48 (2015) Sitobion avenae Olfactory coreceptor gene SaveOrco Feeding Impaired response Fan et al. (2015) Sitobion avenae Lipase maturation factor 2-like gene HIGS Mortality/fecundity Xu et al. (2017) Sitobion avenae Laccase 1 (Lac1) Feeding Mortality Zhang et al. (2018) Sitobion avenae Zinc finger protein (SaZFP) HIGS Mortality/transgenetional gene Sun et al. silencing (2019) Sitobion avenae Ecdysone receptor (EcR) and ultraspiracle Feeding Mortality/fecundity Yan et al. protein (USP) (2016) Sitobion avenae Chitin synthase 1 (CHS1) HIGS Mortality/fecundity Zhao et al. (2018) Pathogens Fusarium Cytochrome P450 lanosterol HIGS Inhibiting fungal mycelium Koch et al. graminearum formation (2013) C-14a-demethylase (CYP51) Fusarium Cytochrome P450 lanosterol C-14a- SIGS Inhibition of fungal growth Koch et al. graminearum demethylase CYP51 (2016) Fusarium Chs3b HIGS Restriction of fungal growth Cheng et al. graminearum through (2015) Blumeria graminis Virulence effector (Avra10) HIGS Reduced fungal development Nowara et al. (2010) Fusarium asiaticum Myosin 5 SIGS Reduced virulence Song et al. (2018) B. graminis f. sp. Ribonuclease-like protein HIGS Reduced virulence Pliego et al. hordei (2013) Ribonuclease-like protein Virulence effector Glucanase Metalloprotease Virulence effector Fusarium culmorum Secreted lipase (Fgl1), Mitogen-activated VIGS and Reduced virulence Chen et al. protein (MAP) kinase (Fmk1), Beta 1,3- HIGS (2016) Glucan synthase (Gls1) Puccinia striiformis Calcineurin homologue (PsCNA1, PsCNB1) VIGS Slower extension of fungal Yin et al. f. sp. tritici hyphae (2010) Puccinia striiformis Mitogen-activated protein kinase (MAPK1), VIGS Reduced virulence Panwar et al. f. sp. tritici Cyclophilin (CYC1), Calcineurin regulatory (2013) subunit (CNB) The Author(s) 2021 aBIOTECH (2021) 2:365–374 369 Table 1 continued Organism Target genes Assay/ Effects References method Puccinia striiformis Protein kinase A catalytic subunit (PsCPK1) VIGS Reduced virulence Qi et al. (2018) f. sp. tritici 3a1/f1 Blumeria Three virulence effectors (SvrPm ) HIGS Reduced virulence Schaefer et al. graminis f. (2020) sp. tritici Puccinia striiformis Glycine-serine-rich effector (PstGSRE1) HIGS Reduced virulence and Qi et al. f. sp. tritici increased H O accumulation (2019a) 2 2 Nematodes Meloidogyne Heat-shock protein 90, isocitrate lyase, HIGS Reduced reproduction Lilley et al. incognita Mi-cpl-1 (2007) Pratylenchus spp. Troponin C (pat-10) Soaking Reduced reproduction Tan et al. solution (2013) Calponin (unc-87) proliferation was significantly reduced together with confer durable broad-spectrum powdery mildew resis- decreasing fungal target gene transcript abundance and tance (Acevedo-Garcia et al. 2017). reduced biomass accumulation in RNAi-based resistant Wheat streak mosaic virus (WSMV) is another per- plants (Panwar et al. 2018). sistent threat to wheat production. Transgenic wheat Powdery mildew caused by Blumeria graminis f. sp. plants constitutively expressing a polycistronic cassette hordei in barley and B. graminis f. sp. tritici in wheat is a of five miR395 arms, known as FanGuard (FGmiR395), serious disease as well. Transgenic barley expressing were exploited to target five distinct regions of the virus dsRNA targeting the avirulence gene Avra10, which genome. The consequent transgenic plants showed corresponds to the resistance gene Mla10, showed nearly complete immunity to WSMV (Fahim et al. 2012). reduced fungal gene transcripts and severely affected In the other case, a segment of 272 bp sequence derived fungal development (Nowara et al. 2010). Silencing of from the coat protein of Triticum mosaic virus (TriMV) 1,3-b-glucanosyltransferase genes (BgGTF1 and was cloned into the hairpin expression vector and BgGTF2) via VIGS that was built on the barley stripe constitutively expressed in wheat. The engineered mosaic virus (BSMV) significantly slowed down the wheat plants showed stable resistance to TriMV (Fahim growth of the powdery mildew fungus (Qi et al. 2019b). et al. 2010). Mildew resistance locus o (Mlo) encodes a transmem- brane protein (Panstruga et al. 2005) that acts as a Management of wheat pests negative regulator to suppress plant immunity in uninfected tissues. It is also involved in protection Several major pests, such as grain aphid (Sitobion avenae), against cell death as well as in responses to biotic and bird cherry-oat aphid (Rhopalosiphum padi), and wheat abiotic stresses (Piffanelli et al. 2002). Down-regulation aphid (Schizaphis graminum), can cause severe yield loss of the TaMlo gene via VIGS resulted in the broad-spec- (Table 1)(Peairs 2008; Smith and Chuang 2014;Yuetal. trum powdery mildew resistance in wheat (Va´rallyay 2014). Transgenic wheat plants expressing a 198 bp et al. 2012). Recently, gene-editing technologies were fragment of dsRNA complementary to the zinc finger used to achieve similar effects. For instance, simulta- protein (SaZFP) of grain aphid can effectively increase its neous knockout of the three TaMlo homoeologues by mortality and reduce its daily fecundity (Sun et al. 2019). TALEN (transcription activator-like effector nuclease) In barley, dsRNA targeting the grain aphid gene encoding produced transgenic wheat plants that were highly salivary sheath protein (SHP), a pivotal component of the resistant to powdery mildew infection, another work stylet penetration process, effectively reduces the repro- produced transgenic wheat plants that carry mutations duction and survival rates of the aphid and the effect can in the TaMLO-A1 allele using the CRISPR-Cas9 technol- be transmitted for seven generations (Abdellatef et al. ogy (Wang et al. 2014). On the other hand, non-trans- 2015). Effects of additional target genes were also con- genic TILLING (Targeting Induced Lesions IN Genomes) firmed by feeding or direct injection into grain aphid, such plants with partial loss-of-function alleles of TaMlo as those encoding catalase, acetylcholinesterase1, The Author(s) 2021 370 aBIOTECH (2021) 2:365–374 cytochrome c oxidase subunit VIIc precursor, and zinc and within 7 days after their addition to aquatic sys- finger protein, and abnormally high mortality and devel- tems containing natural water and various types of opmental stunting were observed (Wang et al. 2015; sediment (Albright et al. 2017; Fischer et al. 2017). Zhang et al. 2013). Despite this, actin-dsRNA derived Colorado potato bee- tle (CPB) remained active for at least four weeks after Management of nematodes in wheat application to potato leaves. It suppressed CPB larval weight gain, delayed its development, and increased its Wheat parasites cause enormous yield losses and mortality (San Miguel and Scott 2016). Therefore, dis- threaten the quality of grains, including Heterodera secting the process of dsRNA degradation is helpful in avenae, H. filipjevi and H. latipons (Table 1) (Toumi and evaluating the potential effect of dsRNA in various Waeyenberge 2013). Targeting of the Ha18764 effector environments and target organisms. protein family genes of H. avenae by the VIGS-based RNAi approach significantly attenuated the parasitism Cost-effective methods for dsRNA production and reproduction status of H. avenae in wheat (Yang et al. 2019). Down-regulation by RNAi of pat-10 and For RNAi application to be practical for field use, the unc-87 genes on Thorne’s meadow nematode (Praty- major hurdle is to produce sufficient amount of dsRNA. lenchus thornei), which infects wheat roots, significantly The traditional dsRNA production method in the labo- reduced the reproduction of the worms (Tan et al. ratory is expensive and produces only a limited amount 2013). Moreover, RNAi in wheat can be stimulated by of dsRNA and thus is not practical for large-scale poly-component biostimulants derived from metabo- application needs (Ahn et al. 2019). Producing dsRNA in lites of various soil streptomycetes which up-regulate bacterial cells with RNaseIII deficiency seems to be an siRNAs and miRNAs in wheat plants. These small RNAs alternative. However, only a handful works have are complementary to cereal cyst nematode mRNA and demonstrated microbial-based dsRNA production. One hence suppress their reproduction providing resistance approach uses L1440-HT115 (DE3) system that has to wheat plants (Blyuss et al. 2019). been successfully applied in the RNAi of Mythimna separate (Das et al. 2015; Parsons et al. 2018; Zhang et al. 2010). With more research underway, the pro- CHALLENGES FOR USING RNAI TECHNOLOGY duction efficiency of this system should be augmented to meet market demands. While the outlook of using RNAi for plant protection appears to be promising, several issues need to be Off-target effects resolved before efficient practical applications. RNAi is a sequence homology-dependent mechanism. The stability of dsRNA Several studies show that siRNA is not always specific and can have off-target effects and thus is problematic One of the primary concerns for the use of RNA as a in disease management (Mamta and Rajam 2017). Some biopesticide is their stability, especially for the spray- target genes are highly conserved between species able dsRNA and siRNA applications. Microorganisms in which increases the likelihood of off-targets among the environment can degrade dsRNA prior to their them. The sequences of vATPaseA and vATPaseEfrom L. uptake by pathogens or pests. Rapid degradation of decemlineata, for instance, shared 83% and 79% dsRNA may occur by nucleases in the saliva, gut lumen, nucleotide-sequence identities to their counterparts in and/or haemolymph of pests as well (Allen and Walker Western Corn Rootworm (WCR), respectively. dsRNAs III 2012; Chung et al. 2018; CoGuan et al. 2018; Katoch from WCR vATPaseA and vATPaseE could reduce the and Thakur 2012; Kennedy et al. 2004; Luo et al. 2013). fitness of Colorado potato beetle (CPB; Leptinotarsa The high or low pH found in the gut lumens of some decemlineata) in a bioassay (Baum et al. 2007). Com- pests can also reduce dsRNA stability either directly or putational design program is needed for specific and indirectly by affecting the activity of gut nucleases systemic evaluation of non-target and off-target effects (Cooper et al. 2019). which should be further verified by additional bioas- Other environmental factors may exert different says. In addition, feeding studies revealed that dsRNAs effects on the stability of dsRNA and sRNA. Several of at least 60 nucleotide (nt) in length are necessary for works show that dsRNA is degraded to unde- an efficient RNAi response in D. virgifera (Bolognesi tectable levels within 48 h after their application on et al. 2012) and Tribolium castaneum (Wang et al. three types of soil (silt loam, loamy sand, and clay loam) 2019). A minimum of 21 nt was required for the size of The Author(s) 2021 aBIOTECH (2021) 2:365–374 371 siRNA for efficient protection against WCR and active lowering costs of the technology. There is no doubt that orthologs (Bachman et al. 2013). a new era of disease control based on RNAi technology for crop protection is right at the corner. RNAi resistance Acknowledgements We are grateful for the financial support by the National Natural Science Foundation of China (No. 31701429), Pests and pathogens can develop resistance to RNAi- National Key Program for Transgenic Research (No. based products through various mechanisms as they do 2016ZX08009-001), and the ‘Yellow Delta Scholarship’ from the for conventional biopesticides. Compared to conven- municipal government of Dongying. tional commercialized transgenic crops expressing Bt Author contributions LM and SSL conceived the topic. S.S.L toxins for pest management (James, 2010). RNAi-based drafted the first manuscript. SFG and ALL read the draft and gave strategy induces down-regulation of the target gene by substantial suggestions for revision, LM revised the manuscript in-complete resistance in most of cases. This may with the help from YBM reduce the selection pressure on the pathogen that may Compliance with ethical standards contribute to durable resistance. But genetic variation in pathogenic organisms may also cause single nucleotide Conflict of interest There are no conflicts of interest. polymorphisms (SNPs) in the target gene. The efficiency of RNAi would be cut down owing to the reduction of Ethical approval We declare that all materials and methods comply with required ethical standards. complementarity between the target gene and the dsRNA. Synonymous SNPs lead to nearly no fitness cost Open Access This article is licensed under a Creative Commons on the pathogens and pests, but the difference between Attribution 4.0 International License, which permits use, sharing, dsRNA and the original gene sequences reduces their adaptation, distribution and reproduction in any medium or for- mat, as long as you give appropriate credit to the original complementarity, causing reduced RNAi effect or RNAi author(s) and the source, provide a link to the Creative Commons resistance (Scott et al. 2013; Yu et al. 2016). Thereby, licence, and indicate if changes were made. The images or other the potential of RNAi resistance should be taken into third party material in this article are included in the article’s consideration in application. Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will CONCLUSIONS AND FUTURE PROSPECTS need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. In the past few years, we have seen diverse applications of RNAi in crop protection methodologies against pests, pathogens, and nematodes. RNAi technology has emerged as a promising new strategy for wheat pro- References tection either. The wide use of HIGS on a commercial scale appears possible soon. The major obstacles for the Abdellatef E, Will T, Koch A, Imani J, Vilcinskas A, Kogel KH (2015) HIGS strategy will be resolved, by optimal target and Silencing the expression of the salivary sheath protein causes fragment selection methods, highly efficient transfor- transgenerational feeding suppression in the aphid Sitobion avenae. 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Journal

aBIOTECHSpringer Journals

Published: Dec 1, 2021

Keywords: Double-stranded RNA; Pathogens; Pests; Nematodes; RNA interference; Small RNA; Wheat

References