Plant miRNAs Research Articles

Genome-Wide Investigation of the MiR166 Family Provides New Insights into Its Involvement in the Drought Stress Responses of Tea Plants (Camellia sinensis (L.) O. Kuntze)

MicroRNA166 (miR166) is a highly conserved plant miRNA that plays a crucial role in plant growth and the resistance to various abiotic stresses. However, the miR166s in tea (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. This study identified 30 mature miR166s and twelve pre-miR166s in tea plants. An evolutionary analysis revealed that csn-miR166s originating from the 3′ arm of their precursors were more conserved than the csn-miR166s derived from the 5′ arm of their precursors. The twelve pre-miR166s in tea were divided into two groups, with csn-MIR166 Scaffold364-2 separated from the other precursors. The Mfold-based predictions indicated that the twelve csn-MIR166s formed typical and stable structures comprising a stem-loop hairpin, with minimum free energy ranging from −110.90 to −71.80 kcal/mol. An analysis of the CsMIR166 promoters detected diverse cis-acting elements, including those related to light responses, biosynthesis and metabolism, abiotic stress defenses, and hormone responses. There was no one-to-one relationship between the csn-miR166s and their targets, but most csn-miR166s targeted HD-Zip III genes. Physiological characterization of tea plants under drought stress showed that leaf water content proportionally decreased with the aggravation of drought stress. In contrast, tea leaves’ malondialdehyde (MDA) content proportionally increased. Moreover, the cleavage site of the ATHB-15-like transcript was identified according to a modified 5′ RNA ligase-mediated rapid amplification of cDNA ends. The RT-qPCR data indicated that the transcription of nine csn-miR166s was negatively correlated with their target gene.

Unveiling the nature's fruit basket to computationally identify Citrus sinensis csi-mir169–3p as a probable plant miRNA against Reference and Omicron SARS-CoV-2 genome

The fundamental role of microRNAs (miRNAs) has long been associated with regulation of gene expression during transcription and post transcription of mRNA's 3′UTR by the RNA interference mechanism. Also, the process of how miRNAs tend to induce mRNA degradation has been predominantly studied in many infectious diseases. In this article, we would like to discuss the interaction of dietary plant miRNAs derived from fresh fruits against the viral genome of the causative agent of COVID-19, specifically targeting the 3′UTR of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) genome. Expanding the analysis, we have also identified plant miRNAs that interact against the Omicron (B.1.1.529) variant of SARS-CoV-2 across 37 countries/territories throughout the world. This cross-species virus-plant interaction led us to identify the alignment of dietary plant miRNAs found in fruits like Citrus sinensis (Orange), Prunus persica (Peaches), Vitis vinifera (Grapes) and Malus domestica (Apple) onto the viral genomes. In particular, the interaction of C. sinensis miRNA - csi-miR169–3p and SARS-CoV-2 is noteworthy, as the targeted 3′UTR region “CTGCCT” is found conserved amongst all curated 772 Omicron variants across the globe. Hence this site “CTGCCT” and miRNA csi-miR169–3p may become promising therapeutic candidates to induce viral genome silencing. Thereby, this study reveals the mechanistic way of how fruits tend to enact a fight against viruses like SARS-CoV-2 and aid in maintaining a strong immune system of an individual.

Advances in the regulation of plant salt-stress tolerance by miRNA.

Salt stress significantly affects the growth, development, yield, and quality of plants. MicroRNAs (miRNAs) are involved in various stress responses via target gene regulation. Their role in regulating salt stress has also received significant attention from researchers. Various transcription factor families are the common target genes of plant miRNAs. Thus, regulating the expression of miRNAs is a novel method for developing salt-tolerant crops. This review summarizes plant miRNAs that mediate salt tolerance, specifically miRNAs that have been utilized in genetic engineering to modify plant salinity tolerance. The molecular mechanism by which miRNAs mediate salt stress tolerance merits elucidation, and this knowledge will promote the development of miRNA-mediated salt-tolerant crops and provide new strategies against increasingly severe soil salinization.

Accurate quantification of 3'-terminal 2'-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR.

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Digestion of Plant Dietary miRNAs Starts in the Mouth under the Protection of Coingested Food Components and Plant-Derived Exosome-like Nanoparticles.

The regulatory functions of plant miRNAs on mammalian bodies are controversial, mainly because stability of the miRNAs in the digestive tract, as the prerequisite for their cross-kingdom effects, has somehow been overlooked. Hence, as the first stage of food ingestion, stability of plant miRNAs in human saliva has been investigated. The results show that plant miRNAs are of considerable resistance against salivary digestion, as surviving miRNAs more than 20 fM are detected. The stability varies dramatically, which can be explained by the difference in tertiary structure, governing their affinities to RNase. Surprisingly, miRNAs of low initial concentrations can end up with high survival rates after digestion. Plant miRNAs can be loaded into exosome-like nanoparticles (ELNs) and microcapsules formed by food components, both of which protect the miRNAs from being degraded in human saliva. Overall, plant miRNAs can apply certain strategies to maintain constant concentrations, paving the way for their potential cross-kingdom effects.

MiR390 family of Cymbidium goeringii is involved in the development of reproductive organs in transgenic Arabidopsis

BackgroundmiR390s is an ancient family with a high level of conservation among plant miRNAs. Through the auxin signal transduction pathway, miR390 participates in diverse biological processes of plant growth and development. As an important Chinese traditional orchid, Cymbidium goeringii has unique flower shape and elegant fragrance. But its development has been greatly restricted because of the low flower bud differentiation and the difficult reproduction. This study aims to provide guidance for the role of cgo-miR390 in reproductive organ development to enhance the ornamental and economic value of Cymbidium.ResultsMIR390a, MIR390b and MIR390c of C. goeringii were cloned, and their length ranged from 130 to 150 nt. Each precursor sequence of cgo-miR390 contains 2 to 3 mature miRNAs. Three kinds of cgo-miR390s displayed distinct temporal and spatial expression patterns during floral development in C. goeringii. The overexpression of MIR390s alters morphology and function of stamens and pistils in Arabidopsis, such as enlargement of anther aspect ratio and separation of stylar and stigmas, which affects the development of fruits and seeds. In particular, the pollen amount decreased and the seed abortion rate increased in cgo-MIR390c-overexpressed plants.Conclusionscgo-miR390 family affected the development of reproductive organs in transgenic Arabidopsis. The study provides references for the genetic improvement for orchid with potentially great economic benefit.

Functional characterization of a 'plant-like' HYL1 homolog in the cnidarian Nematostella vectensis indicates a conserved involvement in microRNA biogenesis.

While the biogenesis of microRNAs (miRNAs) in both animals and plants depends on the RNase III Dicer, its partner proteins are considered distinct for each kingdom. Nevertheless, recent discovery of homologs of Hyponastic Leaves1 (HYL1), a 'plant-specific' Dicer partner, in the metazoan phylum Cnidaria, challenges the view that miRNAs evolved convergently in animals and plants. Here, we show that the HYL1 homolog Hyl1-like a (Hyl1La) is crucial for development and miRNA biogenesis in the cnidarian model Nematostella vectensis. Inhibition of Hyl1La by morpholinos resulted in metamorphosis arrest in Nematostella embryos and a significant reduction in levels of most miRNAs. Further, meta-analysis of morphants of miRNA biogenesis components, like Dicer1, shows clustering of their miRNA profiles with Hyl1La morphants. Strikingly, immunoprecipitation of Hyl1La followed by quantitative PCR revealed that in contrast to the plant HYL1, Hyl1La interacts only with precursor miRNAs and not with primary miRNAs. This was complemented by an in vitro binding assay of Hyl1La to synthetic precursor miRNA. Altogether, these results suggest that the last common ancestor of animals and plants carried a HYL1 homolog that took essential part in miRNA biogenesis and indicate early emergence of the miRNA system before plants and animals separated.

A fungal effector suppresses the nuclear export of AGO1–miRNA complex to promote infection in plants

Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1–microRNA (AGO1–miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAi-dependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens.

The miR159‐MYB33‐ABI5 module regulates seed germination in Arabidopsis

AbstractDrought stress restricts crop productivity and exacerbates food shortages. The plant hormone, abscisic acid (ABA), has been shown to be a pivotal player in the regulation of drought tolerance and seed germination in plants. ABA accumulates under abiotic stresses to promote miR159 expression. miR159 is an ancient and conserved plant miRNA that plays diverse roles in plant development, seed germination, and drought response in Arabidopsis. Our previous studies demonstrated that miR159 regulates the vegetative phase change by repressing the ABI5 activation and thereafter preventing hyperactivation of miR156. However, whether the miR159‐MYB33‐ABI5 module plays a role in seed germination and drought response, and if so, how they interact genetically, remain largely unexplored. Here, we show that loss‐of‐function of miR159 (mir159ab) confers enhanced drought tolerance and hypersensitivity of seed germination to ABA. Genetic analyses demonstrated that loss‐of‐function mutation in the ABI5 gene suppresses the hypersensitivity of mir159ab to ABA, and the insensitivity of myb33 seeds to ABA treatment is ABI5 dependent. ABI5 functions downstream of MYB33 and miR159 in response to ABA. Therefore, our results uncover a new role for the miR159‐MYB33‐ABI5 module in the regulation of drought response and seed germination in plants.

MicroRNA Mediated Plant Responses to Nutrient Stress.

To complete their life cycles, plants require several minerals that are found in soil. Plant growth and development can be affected by nutrient shortages or high nutrient availability. Several adaptations and evolutionary changes have enabled plants to cope with inappropriate growth conditions and low or high nutrient levels. MicroRNAs (miRNAs) have been recognized for transcript cleavage and translational reduction, and can be used for post-transcriptional regulation. Aside from regulating plant growth and development, miRNAs play a crucial role in regulating plant’s adaptations to adverse environmental conditions. Additionally, miRNAs are involved in plants’ sensory functions, nutrient uptake, long-distance root transport, and physiological functions related to nutrients. It may be possible to develop crops that can be cultivated in soils that are either deficient in nutrients or have extreme nutrient supplies by understanding how plant miRNAs are associated with nutrient stress. In this review, an overview is presented regarding recent advances in the understanding of plants’ responses to nitrogen, phosphorus, potassium, sulfur, copper, iron, boron, magnesium, manganese, zinc, and calcium deficiencies via miRNA regulation. We conclude with future research directions emphasizing the modification of crops for improving future food security.

In silico identification of candidate miRNA-encoded Peptides in four Fabaceae species

MicroRNAs (miRNAs) are one of the main regulators of gene expression. Recent studies have demonstrated that primary transcripts of miRNAs (pri-miRNAs) encode regulatory peptides, called miRNA-encoded peptides (miPEPs), capable of enhancing the expression of their associated miRNAs in plants. In this work, we aimed to computationally identify miPEPs produced by small open reading frames (ORFs) in pri-miRNAs from four species of Fabaceae. Five families of miRNAs were investigated, based on their role in plant-microorganism interaction. We used the miR171 family as a training dataset centered on the information about mtr-miPEP171b and vvi-miPEP171d already described. From the sequences of the pri-miRNAs and the genomic regions where they were located, ORFs encoding putative miPEPs were predicted. The 5′-most ORFs encoding peptides on pri-miRNAs were aligned and the amino acids conservation was observed. In total, 81 sequences of potential miPEPs were identified. We found conserved miPEPs inside pri-miRNAs from soybean and between soybean, common bean, and cowpea. Besides, our results showed few conserved miPEPs among isoforms of the same miRNA and no conservation among different miRNA families, which indicate the possible specificity of miPEPs in relation to their corresponding miRNAs. Our findings contribute to the understanding of miPEPs features in plants and provide the basis for studies aiming the biotechnological use of miPEPs in leguminous species.

Small RNA and Degradome Sequencing Reveal Important MicroRNA Function in Nicotiana tabacum Response to Bemisia tabaci.

MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, are key molecules in many biological and metabolic processes of plant growth, development and stress response via targeting mRNAs. The phloem-feeding insect whitefly Bemisia tabaci (Hemiptera, Aleyrodidae) is a serious pest that causes devastating harm to agricultural production worldwide. However, the function of host miRNAs in the response to whitefly infestation remains unclear. Here, we sequenced the small RNA and degradome of tobacco (Nicotiana tabacum L.), after and before infestation by B. tabaci. We identified 1291 miRNAs belonging to 138 miRNA families including 706 known miRNAs and 585 novel miRNAs. A total of 47 miRNAs were differentially expressed, of which 30 were upregulated and 17 were downregulated by whitefly exposure. Then, computational analysis showed that the target genes of differential miRNAs were involved in R gene regulation, plant innate immunity, plant pathogen defense, the plant hormone signal pathway and abiotic stress tolerance. Furthermore, degradome analysis demonstrated that 253 mRNAs were cleaved by 66 miRNAs. Among them, the targets cleaved by upregulated miR6025, miR160, miR171, miR166 and miR168 are consistent with our prediction, suggesting that pathogen-related miRNAs may function in plant defense against whitefly. Moreover, our results show that plant miRNA response and miRNA-mediated post-transcriptional regulation for phloem-feeding insect infestation are similar to pathogen invasion. Our study provides additional data to further elucidate how host plants respond and defend the phloem-feeding insects.

Advances in the research of microRNA in Orchidaceae

As a class of small non-coding RNAs, microRNA (miRNA) is widely present and plays important regulatory roles in plant growth, development and stress response. Based on the mechanism of miRNAs in plants, we review the identification of miRNAs in some genera of Orchidaceae, the specific functions of several miRNAs and other relevant studies on miRNAs in the last decade, in order to provide a reference for better understanding function and regulatory network of small RNAs in orchids.

Profiling of Single-cell-type-specific MicroRNAs in Arabidopsis Roots by Immunoprecipitation of Root Cell-layer-specific GFP-AGO1.

MicroRNAs (miRNA) are small (21-24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR ) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves. This protocol was validated in: Dev Cell (2022), DOI: 10.1016/j.devcel.2022.03.015 Graphical abstract.

Time-dependent oscillation of pre-MiRNAs, mature MiRNAs and MiRNA targets in tomato plants subjected to varying abiotic stress conditions

Miniature non-coding RNAs known as MicroRNAs (MiRNA) are transcribed from their independent genes. MiRNAs bind to and regulate various transcripts. They have been linked to a number of biological processes ranging from organ growth, senescence, and pathogen response. We used model crop tomato to study how miRNA genes respond to a variety of abiotic stress, e.g. salt, drought and cold temperature. Precursor-miRNA (pre-miR) levels of six highly conserved plant miRNAs were monitored over 24 h time period. Expression of pre-miR159, −160, −162 and −397 were induced, while pre-miR171a and −319 levels were down regulated as a reaction to a variety of stress treatments. The expression profiles related to miRNA precursors exhibit unique expression pattern to the stress subjected. We followed expression of three mature miRNAs, namely, miR159, miR160 and miR171 upon stress treatment and these exhibited expression profiles similar to their corresponding precursors. Moreover, modulation in mature miRNA levels affected expression of their target transcripts. Prediction of promoter sequences of various tomato pre-miRNAs revealed several cis-elements that may have functional consequence on the gene expression as a consequence of stress. Together, these findings indicate that miRNA genes are sensitive to stress induced due to abiotic factors (climatic stress caused due to drought and cold as well as salt). They demonstrated drastic expression changes to most of the stress examined, a strategy to fine tune transcriptome in a time-efficient manner.

Possible Functions of the Conserved Peptides Encoded by the RNAprecursors of miRNAs in Plants

MicroRNAs (miRNAs) are short double-stranded regulatory molecules derived from precursor transcripts called primary miRNAs (pri-miRNAs) and transcribing in the nucleus from chromosomal DNA by the RNA polymerase II.

Molecular Manipulation of the miR396 and miR399 Expression Modules Alters the Response of Arabidopsis thaliana to Phosphate Stress.

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.

Insights into Health-Promoting Effects of Plant MicroRNAs: A Review.

Plant-derived microRNAs (miRNAs) play a significant role in human health and are "dark nutrients", as opposed to traditional plant nutrients, as well as important components of food diversification. Studies have revealed that multiple plant-derived miRNA pathways affect human health. First, plant miRNAs regulate plant growth and development and accumulation of metabolites, which alters the food quality and thus indirectly interferes with the health of the host. Moreover, when absorbed in vivo, some miRNAs may target the host cell mRNAs to affect protein expression. In addition, plant miRNAs target and reshape the human gut microbiota (GM), which interferes with the physiology and metabolism of the host. Therefore, miRNAs play a significant role in the cross-kingdom communication of plants, GM, and the host and in maintaining a balance of the three. Future contributions of plant miRNAs can bring new perspectives and opportunities to better understand food nutrition and health care research, which will facilitate the right exploitation of plant resources.

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

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.