Plant-Generated Artificial Small RNAs Mediated Aphid Resistance

PLOS ONE, Dec 2019

Background RNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects. Methodology/Principal findings In this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA. Conclusions/Significance Our results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.

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Plant-Generated Artificial Small RNAs Mediated Aphid Resistance

Citation: Guo H, Song X, Wang G, Yang K, Wang Y, et al. ( Plant-Generated Artificial Small RNAs Mediated Aphid Resistance Hongyan Guo 0 Xiaoguang Song 0 Guiling Wang 0 Kun Yang 0 Yu Wang 0 Libo Niu 0 Xiaoying Chen 0 Rongxiang Fang 0 Biao Ding, The Ohio State University, United States of America 0 1 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences , Beijing , China , 2 National Plant Gene Research Center , Beijing , China , 3 University of Chinese Academy of Sciences , Beijing , China , 4 School of Life Science and Technology, University of Electronic Science and Technology of China , Chengdu , China Background: RNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insectresistant plants by expressing hpRNAs targeting essential genes of insects. Methodology/Principal findings: In this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2specific hpRNA. Conclusions/Significance: Our results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects. - Funding: This work was supported by grant KSCX2-EW-N-06-3 from the Chinese Academy of Sciences (http://www.cas.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The phenomenon of RNA silencing was first discovered during plant transgenic studies where it was termed co-suppression [1,2]. Although it has various terms, post-transcriptional gene silencing (PTGS) in plants, RNA interference (RNAi) in animals and quelling in fungi, RNA silencing is based on a highly conserved mechanistic frame [13], and is a natural regulatory mechanism acting against genomic intruders and modulating endogenous gene expression in eukaryotes [47]. Because RNA silencing can be exploited to regulate gene expression through knock-down of the nucleotide sequence-matched target transcripts, it has become an effective reverse genetics approach in functional genomics and a powerful tool to develop transgenic plants that have enhanced resistance against diseases caused by e.g. virus pathogens or insect pests [813]. Earlier works on virus-resistant transgenic plants were based on pathogen-derived resistance (PDR) which was mediated by expression of viral sequences coding for, e.g. coat protein or RNA-dependent RNA-polymerase of RNA viruses or replicationassociated protein of DNA viruses [1416]. Even though the underlying mechanisms are not fully understood, both proteinand RNA-mediated interferences are thought to co-exist, and these strategies have been extensively used as tools to engineer virus-resistant transgenic plants [17]. As the RNA silencing mechanisms were demonstrated and studied intensively, many studies focused on the application of these RNA-mediated strategies to generate transgenic plants that are resistant to virus V-type proton ATPase subunit E-like RR1 cuticle protein 1 (cprr1-1) 40S ribosomal protein S5-like isoform-1 tubulin folding cofactor D (TBCD) ribosomal protein S14 (Rps14) mediator complex subunit 31 (Med31) SWI/SNF-related matrix-associated actin-dependen (...truncated)


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Hongyan Guo, Xiaoguang Song, Guiling Wang, Kun Yang, Yu Wang, Libo Niu, Xiaoying Chen, Rongxiang Fang. Plant-Generated Artificial Small RNAs Mediated Aphid Resistance, PLOS ONE, 2014, 5, DOI: 10.1371/journal.pone.0097410