Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice

Zhang, Dandan; Zhang, Nannan; Shen, Wenzhong; Li, Jianfeng

doi:10.3390/ijms20225620

Cited by 11 publications

(8 citation statements)

References 59 publications

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“…Total RNA was isolated from BPH female adults or rice using the TRIzol reagent (Invitrogen, CA, USA). Regular or stem-loop RT-qPCR was performed as previously described [ 51 ]. Briefly, total RNA was converted into the first-strand cDNA with stem-loop RT primers for osa-miR162a or osa-miR162a-m1 or with an oligo dT primer for other genes using the PrimeScript RT reagent Kit plus genomic DNA Eraser (TaKaRa, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development

Shen

Cao

Liu

et al. 2021

IJMS

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Rice is a main food crop for more than half of the global population. The brown planthopper (BPH, Nilaparvata lugens) is one of the most destructive insect pests of rice. Currently, repeated overuse of chemical insecticides represents a common practice in agriculture for BPH control, which can induce insect tolerance and provoke environmental concerns. This situation calls for innovative and widely applicable strategies for rice protection against BPH. Here we report that the rice osa-miR162a can mediate cross-kingdom RNA interference (RNAi) by targeting the NlTOR (Target of rapamycin) gene of BPH that regulates the reproduction process. Through artificial diet or injection, osa-miR162a mimics repressed the NlTOR expression and impaired the oviposition of BPH adults. Consistently, overproduced osa-miR162a in transgenic rice plants compromised the fecundity of BPH adults fed with these plants, but meanwhile perturbed root and grain development. To circumvent this issue, we generated osa-miR162a-m1, a sequence-optimized osa-miR162a, by decreasing base complementarity to rice endogenous target genes while increasing base complementarity to NlTOR. Transgenic overexpression of osa-miR162a-m1 conferred rice resistance to BPH without detectable developmental penalty. This work reveals the first cross-kingdom RNAi mechanism in rice-BPH interactions and inspires a potentially useful approach for improving rice resistance to BPH. We also introduce an effective strategy to uncouple unwanted host developmental perturbation from desirable cross-kingdom RNAi benefits for overexpressed plant miRNAs.

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Section: Methodsmentioning

confidence: 99%

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development

Shen

Cao

Liu

et al. 2021

IJMS

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“…It has contributed to the dissection of signaling pathways in responses to plant hormones or environmental factors ( Hwang and Sheen, 2001 ; Yoo et al, 2007 ; Li et al, 2019 ; Lehmann et al, 2020 ). Recently, knockdown approaches using either RNAi or artificial microRNA (amiRNA) have enabled a reduction in the expression of the endogenous target gene in protoplasts, extending the application of this technology to evaluate gene knockdown effects ( Ossowski et al, 2008 ; Kim and Somers, 2010 ; Zhang et al, 2019 ; Vachova et al, 2020 ). Furthermore, the protoplast viability extension has enabled the investigation of long-term kinetic molecular responses using a luciferase-based reporter for circadian biology ( Kim and Somers, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts

Doan

Kim

2022

Front. Plant Sci.

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Leaf senescence is the final stage of leaf development preceding death, which involves a significant cellular metabolic transition from anabolism to catabolism. Several processes during leaf senescence require coordinated regulation by senescence regulatory genes. In this study, we developed a rapid and systematic cellular approach to dissect the functional roles of genes in senescence regulation through their transient expression in Arabidopsis protoplasts. We established and validated this system by monitoring the differential expression of a luciferase-based reporter that was driven by promoters of SEN4 and SAG12, early and late senescence-responsive genes, depending on effectors of known positive and negative senescence regulators. Overexpression of positive senescence regulators, including ORE1, RPK1, and RAV1, increased the expression of both SEN4- and SAG12-LUC while ORE7, a negative senescence regulator decreased their expression. Consistently with overexpression, knockdown of target genes using amiRNAs resulted in opposite SAG12-LUC expression patterns. The timing and patterns of reporter responses induced by senescence regulators provided molecular evidence for their distinct kinetic involvement in leaf senescence regulation. Remarkably, ORE1 and RPK1 are involved in cell death responses, with more prominent and earlier involvement of ORE1 than RPK1. Consistent with the results in protoplasts, further time series of reactive oxygen species (ROS) and cell death assays using different tobacco transient systems reveal that ORE1 causes acute cell death and RPK1 mediates superoxide-dependent intermediate cell death signaling during leaf senescence. Overall, our results indicated that the luciferase-based reporter system in protoplasts is a reliable experimental system that can be effectively used to examine the regulatory roles of Arabidopsis senescence-associated genes.

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“…Many plant monocistronic MIRNA genes (monocistronic in this context indicates the presence of one single MIRNA hairpin) have been used to express amiRNAs in dicots and monocots, including ath‐MIR159 (Niu et al , 2006), tae‐MIR164 (Gasparis et al , 2017), ath‐MIR167b , ath‐MIR171a (Ai et al , 2011; Qu et al , 2007), ath‐MIR172a (Schwab et al , 2006), ath‐MIR319a (Jover‐Gil et al , 2014; Schwab et al , 2006), ath‐MIR390a (Carbonell et al , 2014), osa‐MIR390 (Carbonell et al , 2015), ath‐MIR395a (Liang et al , 2012) and osa‐MIR528 (Warthmann et al , 2008). Amongst these, the ath‐MIR319a and osa‐MIR528 backbones have been used most commonly because they are highly conserved across the plant kingdom (Zhang et al , 2019). Recently, the ath‐MIR390a foldback was demonstrated to be very efficient, expressing significantly higher levels of amiRNAs than ath‐MIR319a in Nicotiana benthamiana (Carbonell et al , 2014).…”

Section: Introductionmentioning

confidence: 99%

Expression and processing of polycistronic artificial microRNAs and trans‐acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana

2021

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Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach, as they have the advantage of producing just a single functional small RNA, which can be designed for high target specificity and low off-target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans-acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA-and tasiRNA-based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. All analyses used transiently expressed transgenes delivered by infiltration of leaves with Agrobacterium tumefacians. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5 0 -most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3 0 -end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single-target assays such as RNA blots or real-time polymerase chain reaction, is also discussed.

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Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice

Cited by 11 publications

References 59 publications

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development

Overexpression of an Osa-miR162a Derivative in Rice Confers Cross-Kingdom RNA Interference-Mediated Brown Planthopper Resistance without Perturbing Host Development

Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts

Expression and processing of polycistronic artificial microRNAs and trans‐acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana

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