Improving cassava bacterial blight resistance by editing the epigenome.

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

Hiding in plain sight: New virus genomes discovered via a systematic analysis of fungal public transcriptomes.

The distribution and diversity of RNA viruses in fungi is incompletely understood due to the often cryptic nature of mycoviral infections and the focused study of primarily pathogenic and/or economically important fungi. As most viruses that are known to infect fungi possess either single-stranded or double-stranded RNA genomes, transcriptomic data provides the opportunity to query for viruses in diverse fungal samples without any a priori knowledge of virus infection. Here we describe a systematic survey of all transcriptomic datasets from fungi belonging to the subphylum Pezizomycotina. Using a simple but effective computational pipeline that uses reads discarded during normal RNA-seq analyses, followed by identification of a viral RNA-dependent RNA polymerase (RdRP) motif in de novo assembled contigs, 59 viruses from 44 different fungi were identified. Among the viruses identified, 88% were determined to be new species and 68% are, to our knowledge, the first virus described from the fungal species. Comprehensive analyses of both nucleotide and inferred protein sequences characterize the phylogenetic relationships between these viruses and the known set of mycoviral sequences and support the classification of up to four new families and two new genera. Thus the results provide a deeper understanding of the scope of mycoviral diversity while also increasing the distribution of fungal hosts. Further, this study demonstrates the suitability of analyzing RNA-seq data to facilitate rapid discovery of new viruses.

Functional dissection of the ARGONAUTE7 promoter.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis thaliana has 10 ARGONAUTE (AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1,AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggered TAS3 pathway functions in timing and polarity.

Antiviral ARGONAUTEs Against Turnip Crinkle Virus Revealed by Image-Based Trait Analysis.

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis (Arabidopsis thaliana) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

Raspberry Pi-powered imaging for plant phenotyping.

PREMISE OF THE STUDY: Image-based phenomics is a powerful approach to capture and quantify plant diversity. However, commercial platforms that make consistent image acquisition easy are often cost-prohibitive. To make high-throughput phenotyping methods more accessible, low-cost microcomputers and cameras can be used to acquire plant image data. METHODS AND RESULTS: We used low-cost Raspberry Pi computers and cameras to manage and capture plant image data. Detailed here are three different applications of Raspberry Pi-controlled imaging platforms for seed and shoot imaging. Images obtained from each platform were suitable for extracting quantifiable plant traits (e.g., shape, area, height, color) en masse using open-source image processing software such as PlantCV. CONCLUSIONS: This protocol describes three low-cost platforms for image acquisition that are useful for quantifying plant diversity. When coupled with open-source image processing tools, these imaging platforms provide viable low-cost solutions for incorporating high-throughput phenomics into a wide range of research programs.

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses.

Mitigation of cassava mosaic disease (CMD) focuses on the introgression of resistance imparted by the polygenic recessive (CMD1), dominant monogenic (CMD2) and CMD3 loci. The mechanism(s) of resistance they impart, however, remain unknown. Two CMD susceptible and nine CMD resistant cassava genotypes were inoculated by microparticle bombardment with infectious clones of African cassava mosaic virus Cameroon strain (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Genotypes carrying the CMD1 (TMS 30572), CMD2 (TME 3, TME 204 and Oko-iyawo) and CMD3 (TMS 97/0505) resistance mechanisms showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7days post inoculation (dpi). In contrast, all genotypes initially developed severe CMD symptoms and accumulated high virus titers after inoculation with EACMV KE2 (K201). Resistant genotypes recovered to become asymptomatic by 65dpi with no detectable virus in newly formed leaves. Genotype TMS 97/2205 showed highest resistance to EACMV KE2 (K201) with <30% of inoculated plants developing symptoms followed by complete recovery by 35dpi. Deep sequencing of small RNAs confirmed production of 21-24 nt virus derived small RNAs (vsRNA) that mapped to cover the entire ACMV-CM and EACMV KE2 (K201) viral genomes in both polarities, with hotspots seen within gene coding regions. In resistant genotypes, total vsRNAs were most abundant at 20 and 35dpi but reduced significantly upon recovery from CMD. In contrast, CMD susceptible genotypes displayed abundant vsRNAs throughout the experimental period. The percentage of vsRNAs reads ranked by class size were 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied. The number of vsRNA reads directly correlated with virus titer and CMD symptoms.

Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.

Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism.

Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants.

Artificial small RNAs (sRNAs) are short ≈21-nt non-coding RNAs engineered to inactivate sequence complementary RNAs. In plants, they have been extensively used to silence cellular transcripts in gene function analyses and to target invading RNA viruses to induce resistance. Current artificial sRNA-based antiviral resistance in plants is mainly limited to a single virus, and is jeopardized by the emergence of mutations in the artificial sRNA target site or by the presence of co-infecting viruses. Hence, there is a need to further develop the artificial sRNA approach to generate more broad and durable antiviral resistance in plants. A recently developed toolbox allows for the time and cost-effective large-scale production of artificial sRNA constructs in plants. The toolbox includes the P-SAMS web tool for the automated design of artificial sRNAs, and a new generation of artificial microRNA and synthetic trans-acting small interfering RNA (syn-tasiRNA) vectors for direct cloning and high expression of artificial sRNAs. Here we describe how the simplicity and high-throughput capability of these new technologies should accelerate the study of artificial sRNA-based antiviral resistance in plants. In particular, we discuss the potential of the syn-tasiRNA approach as a promising strategy for developing more effective, durable and broad antiviral resistance in plants.

P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design.

SUMMARY: The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into 'B/c' compatible vectors. AVAILABILITY AND IMPLEMENTATION: The P-SAMS website is available at http://p-sams.carringtonlab.org. CONTACT: acarbonell@ibmcp.upv.es or nfahlgren@danforthcenter.org.

Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis.

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer-preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)-mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild-type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2-type varieties TME 3 and TME 7, but the CMD1-type cultivar TMS 30572 and the CMD3-type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2-mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field-level resistance in CMD2-type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

CG gene body DNA methylation changes and evolution of duplicated genes in cassava.

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

Antiviral roles of plant ARGONAUTES.

ARGONAUTES (AGOs) are the effector proteins functioning in eukaryotic RNA silencing pathways. AGOs associate with small RNAs and are programmed to target complementary RNA or DNA. Plant viruses induce a potent and specific antiviral RNA silencing host response in which AGOs play a central role. Antiviral AGOs associate with virus-derived small RNAs to repress complementary viral RNAs or DNAs, or with endogenous small RNAs to regulate host gene expression and promote antiviral defense. Here, we review recent progress towards understanding the roles of plant AGOs in antiviral defense. We also discuss the strategies that viruses have evolved to modulate, attenuate or suppress AGO antiviral functions.

A Versatile Phenotyping System and Analytics Platform Reveals Diverse Temporal Responses to Water Availability in Setaria.

Phenotyping has become the rate-limiting step in using large-scale genomic data to understand and improve agricultural crops. Here, the Bellwether Phenotyping Platform for controlled-environment plant growth and automated multimodal phenotyping is described. The system has capacity for 1140 plants, which pass daily through stations to record fluorescence, near-infrared, and visible images. Plant Computer Vision (PlantCV) was developed as open-source, hardware platform-independent software for quantitative image analysis. In a 4-week experiment, wild Setaria viridis and domesticated Setaria italica had fundamentally different temporal responses to water availability. While both lines produced similar levels of biomass under limited water conditions, Setaria viridis maintained the same water-use efficiency under water replete conditions, while Setaria italica shifted to less efficient growth. Overall, the Bellwether Phenotyping Platform and PlantCV software detected significant effects of genotype and environment on height, biomass, water-use efficiency, color, plant architecture, and tissue water status traits. All ∼ 79,000 images acquired during the course of the experiment are publicly available.

Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection.

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors.

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.

ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis.

Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.

Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo.

The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart. Importance: To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.