Artificial microRNA Research Articles

CmMLO17 and its partner CmKIC potentially support Alternaria alternata growth in Chrysanthemum morifolium

The Mildew Resistance Locus O (MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemum MLO genes. We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection. Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A. alternata infection. Genes in the abscisic acid (ABA) and Ca2+ signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants. We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca2+ signaling pathways, resulting in reduced susceptibility of chrysanthemum to A. alternata infection. In addition, a candidate gene, CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus. CmKIC overexpression increased the susceptibility of chrysanthemum to A. alternata, whereas CmKIC silencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together in C. morifolium to support the growth of A. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

A virus-derived siRNA activates plant immunity by interfering with ROS scavenging

Virus-derived small interference RNAs (vsiRNAs) not only suppress virus infection in plants via induction of RNA silencing but also enhance virus infection by regulating host defensive gene expression. However, the underlying mechanisms that control vsiRNA-mediated host immunity or susceptibility remain largely unknown. In this study, we generated several transgenic wheat lines using four artificial microRNA expression vectors carrying vsiRNAs from Wheat yellow mosaic virus (WYMV) RNA1. Laboratory and field tests showed that two transgenic wheat lines expressing amiRNA1 were highly resistant to WYMV infection. Further analyses showed that vsiRNA1 could modulate the expression of a wheat thioredoxin-like gene (TaAAED1), which encodes a negative regulator of reactive oxygen species (ROS) production in the chloroplast. The function of TaAAED1 in ROS scavenging could be suppressed by vsiRNA1 in a dose-dependent manner. Furthermore, transgenic expression of amiRNA1 in wheat resulted in broad-spectrum disease resistance to Chinese wheat mosaic virus, Barley stripe mosaic virus, and Puccinia striiformis f. sp. tritici infection, suggesting that vsiRNA1 is involved in wheat immunity via ROS signaling. Collectively, these findings reveal a previously unidentified mechanism underlying the arms race between viruses and plants.

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

Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach; 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 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'-most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3' 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 PCR, is also discussed.

Artificial microRNA derived from the precursors of Ananas comosus, Arabidopsis thaliana, and Oryza sativa effectively silences endogenous genes in MD2 pineapple

Artificial microRNA (amiRNA) is considered the next generation of gene silencing vectors because it can be custom designed to silence any gene of interest in an organism. In the amiRNA mechanism, the precursor microRNA (pre-miRNA) plays an important role in transporting and ensuring that the amiRNA is processed through the endogenous miRNA biogenesis pathway, allowing for the amiRNA to be expressed and function as a gene silencing tool. However, the efficiency of expressing amiRNA between miRNA precursors and plant species varies, as there are no universal precursors that are suitable for use in all species. We therefore attempted to identify precursors that are compatible and efficient for use in the MD2 pineapple with the eventual purpose of studying gene function in the crop. The results showed that three endogenous precursors (aco-MIR156, aco-MIR399 and aco-MIR2673 of Ananas comosus) and two exogenous precursors (ath-MIR319 of Arabidopsis thaliana and osa-MIR528 of Oryza sativa) were compatible in the MD2 pineapple, all of which expressed amiRNA. However, the efficiency of these precursors differed, with the exogenous precursor ath-MIR319 found to be more efficient, that is, produced a higher level of amiRNA than the endogenous precursor itself. The endogenous precursors were then structurally modified to increase their efficiency. This resulted in precursor aco-MIR156 (with a modified stem structure) expressing the highest level of amiRNA. In conclusion, we identified suitable precursors for the amiRNA backbone in pineapple and showed that aco-MIR156 from pineapple was as equally efficient as established precursors, ath-MIR319 and osa-miR528 derived from Arabidopsis thaliana and Oryza sativa, respectively. This result opens the door to designing a set of precursors which are reusable as a backbone for amiRNA silencing in other non-model species. We recommend this newly developed pineapple amiRNA as a tool in gene silencing as it mimics the endogenous miRNA with high efficiency.

Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs.

BackgroundNF-κB is a sequence-specific DNA-binding transcription factor that plays key roles in inflammation and cancer. It is well known that NF-κB is over-activated in these diseases. NF-κB inhibitors are therefore developed as promising drugs for these diseases. However, finding NF-κB inhibitors is dependent on effective screening platforms.MethodsFor providing an easy and visualizable tool for screening NF-κB inhibitors, and other NF-κB-related studies, this study edited all five genes of NF-κB family (RELA, RELB, CREL, NF-κB1, NF-κB2) in three different cell lines (293T, HepG2, and PANC1) with both TALEN and CRISPR. The edited NF-κB genes were repaired by homology-dependent repair using a linear homologous donor containing ZsGreen coding sequence. The edit efficiency was thus directly evaluated by detecting cellular fluorescence. The editing efficiency was also confirmed by PCR detection of NF-κB-ZsGreen fused genes.ResultsIt was found that all genes were more efficiently edited by TALEN in all cells than CRISPR. The positive cells were then isolated from the TALEN-edited cell pool by flow cytometry. The purified positive cells were finally evaluated by regulating NF-κB activity with a known NF-κB inhibitor, BAY 11–7082, and an NF-κB-targeting artificial microRNA, miR533. The results revealed that all the labeled NF-κB genes responded well to the two kinds of NF-κB activity regulators in all cell lines.ConclusionThis study thus obtained 15 cell lines with NF-κB-ZsGreen fused genes, which provide an easy and visualizable tool for screening NF-κB inhibitors and other NF-κB-related studies.

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetic screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

Improvement of host-induced gene silencing efficiency via polycistronic-tRNA-amiR expression for multiple target genes and characterization of RNAi mechanism in Mythimna separata.

SummaryHost‐induced gene silencing (HIGS) emerged as a new strategy for pest control. However, RNAi efficiency is reported to be low in Lepidoptera, which are composed of many important crop pests. To address this, we generated transgenic plants to develop HIGS effects in a maize pest, Mythimna separata (Lepidoptera, Noctuidae), by targeting chitinase encoding genes. More importantly, we developed an artificial microRNA (amiR) based PTA (polycistronic‐tRNA‐amiR) system for silencing multiple target genes. Compared with hpRNA (hairpin RNA), transgenic expression of a PTA cassette including an amiR for the gut‐specific dsRNA nuclease gene MsREase, resulted in improved knockdown efficiency and caused more pronounced developmental abnormalities in recipient insects. When target gene siRNAs were analysed after HIGS and direct dsRNA/siRNA feeding, common features such as sense polarity and siRNA hotspot regions were observed, however, they differed in siRNA transitivity and major 20‐24nt siRNA species. Core RNAi genes were identified in M. separata, and biochemical activities of MsAGO2, MsSID1 and MsDcr2 were confirmed by EMSA (electrophoretic mobility shift assay) and dsRNA cleavage assays, respectively. Taken together, we provide compelling evidence for the existence of the RNAi mechanism in M. separata by analysis of both siRNA signatures and RNAi machinery components, and the PTA system could potentially be useful for future RNAi control of lepidopteran pests.

Artificial microRNA-Based RNA Interference and Specific Gene Silencing for Developing Insect Resistance in Solanum lycopersicum

RNA Interference (RNAi), which works against invading nucleic acids or modulates the expression of endogenous genes, is a natural eukaryotic regulating system, and it works by noncoding smaller RNA molecules. Plant-mediated gene silencing through RNAi can be used to develop plants with insect tolerance at transcriptional or post-transcriptional levels. In this study, we selected Myzus persicae’s acetylcholinesterase 1 gene (Ace 1) as a silencing target to develop transgenic Solanum lycopersicum L. plants’ resistance to aphids. An RNAi plasmid vector containing an artificial microRNA (amiRNA) sequence was engineered and successfully transformed into Jamila and Tomaland, two elite tomato cultivars. A northern blot analysis and PCR were carried out to check the efficacy of Agrobacterium-mediated transformation in T0 transgenic plants. The quantitative PCR data showed a substantial downregulation of the Ace 1 gene in aphids fed in clip cages on T1 transgenic plants. Furthermore, there was a substantial drop in aphid colonies that were fed on T1 transgenic plants of both the cultivars. These findings strongly suggest that transgenic plants that express amiRNA could be an important tool for engineering plants resistant to aphids and possibly for the prevention of viral disease in other plant-infested pests.

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in Nicotiana benthamiana. We engineered sonchus yellow net rhabdovirus (SYNV)-based gene silencing vectors through expressing a sense, antisense, or double-stranded RNAs of target genes. Robust target gene silencing was also achieved with an SYNV vector expressing a designed artificial microRNA. In addition, ectopic expression of a short tandem target mimic RNA using the SYNV vector led to a significant depletion of the target miR165/166 and caused abnormal leaf development. More importantly, SYNV was able to harbor two expression cassettes that permitted simultaneous RNA silencing and overexpression of large reporter gene. This dual capacity vector also enabled systemic expression of a whole-molecule monoclonal antibody consisting of light and heavy chains. These results highlight the utility of the SYNV vector system in gene function studies and agricultural biotechnology and provide a technical template for developing similar vectors of other economically important plant rhabdoviruses.

Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing.

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.

RNA-Based Technologies for Engineering Plant Virus Resistance.

In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.

Oocyte-specific gene knockdown by intronic artificial microRNAs driven by Zp3 transcription in mice.

Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.

Downregulating VAC14 in Guard Cells Causes Drought Hypersensitivity by Inhibiting Stomatal Closure.

Stomata are a key land plant innovation that permit the regulation of gaseous exchanges between the plant interior and the surrounding environment. By opening or closing, stomata regulate transpiration of water though the plant; and these actions are coordinated with acquisition of CO2 for photosynthesis. Stomatal movement is controlled by various environmental and physiological factors and associates with multiple intracellular activities, among which the dynamic remodeling of vacuoles plays a crucial role. Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is critical for dynamic remodeling of vacuoles. Its production requires a PI(3,5)P2-metabolizing complex consisting of FAB1/PIKfyve kinases, SAC phosphatases, and the scaffolding protein VAC14. Although genetic or pharmacological downregulation of PI(3,5)P2 causes hyposensitivity to ABA-induced stomatal closure, whether the effect of PI(3,5)P2 on stomatal movement is cell-autonomous and the physiological consequences of its reduction were unclear. We report that downregulating Arabidopsis VAC14 specifically in guard cells by artificial microRNAs (amiR-VAC14) results in enlarged guard cells and hyposensitivity to ABA- and dark-induced stomatal closure. Vacuolar fission during stomatal closure is compromised by downregulating VAC14 in guard cells. Exogenous application of PI(3,5)P2 rescued the amiR-VAC14 phenotype whereas PI(3,5)P2 inhibitor YM201636 caused wild-type plants to have inhibited stomatal closure. We further show that downregulating VAC14 specifically in guard cells impairs drought tolerance, suggestive of a key role of guard cell-produced PI(3,5)P2 in plant fitness.

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Clay nanosheet-mediated delivery of recombinant plasmids expressing artificial miRNAs via leaf spray to prevent infection by plant DNA viruses

Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants, such as tomato, papaya, cotton, and tobacco. Tomato yellow leaf curl virus (TYLCV) is a typical monopartite begomovirus that has been extensively studied, but methods that can efficiently control begomoviruses are still scarce. In this study, we combined artificial microRNA (amiRNA)-mediated silencing technology and clay nanosheet-mediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants. We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts. Three plant expression vectors expressing pre-amiRNAs were constructed, and recombinant plasmid DNAs (pDNAs) were loaded onto nontoxic and degradable layered double hydroxide (LDH) clay nanosheets. LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves. We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying, while the pDNAs were sustainably detected for 35 days after the spray, suggesting that the LDH nanosheets released pDNAs in a sustained manner, protected pDNAs from degradation and efficiently delivered pDNAs into plant cells. Importantly, when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV, both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days, while the levels of H2O2 were significantly increased in those plants. Taken together, these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.

Host-derived artificial miRNA-mediated silencing of ecdysone receptor gene provides enhanced resistance to Helicoverpa armigera in tomato

Helicoverpa armigera causes huge crop losses due to its polyphagous nature. The present study demonstrates the use of artificial microRNA (amiRNA) mediated gene silencing approach to generate insect resistant tomato plants. Ecdysone receptor (HaEcR) gene of the target pest, H. armigera, which is involved in the regulation of all developmental stages of the insect life cycle, was silenced by sequence-specific amiRNA (amiRNA-HaEcR). Continuous feeding on detached tomato leaves expressing the amiRNA-319a-HaEcR resulted in reduced target gene transcripts and affected the overall growth and survival of H. armigera. Not only the target gene was down-regulated but, the feeding also affected the expression of down-stream genes involved in the ecdysone signaling pathway. The resistant trait was also observed in T1 generation of tomato transgenic lines. These results further established the role of EcR as a master regulator in insect development and effectiveness of amiRNA technology for efficient control of H. armigera.

The Arabidopsis KH-domain protein FLOWERING LOCUS Y delays flowering by upregulating FLOWERING LOCUS C family members.

We identified FLY as a previously uncharacterized RNA-binding-family protein that controls flowering time by positively regulating the expression of FLC clade members. The ability of flowering plants to adjust the timing of the floral transition based on endogenous and environmental signals contributes to their adaptive success. In Arabidopsis thaliana, the MADS-domain protein FLOWERING LOCUS C (FLC) and the FLC clade members FLOWERING LOCUS M/MADS AFFECTING FLOWERING1 (FLM/MAF1), MAF2, MAF3, MAF4, and MAF5 form nuclear complexes that repress flowering under noninductive conditions. However, how FLM/MAF genes are regulated requires further study. Using a genetic strategy, we showed that the previously uncharacterized K-homology (KH) domain protein FLOWERING LOCUS Y (FLY) modulates flowering time. The fly-1 knockout mutant and FLY artificial microRNA knockdown line flowered earlier than the wild type under long- and short-day conditions. The knockout fly-1 allele, a SALK T-DNA insertion mutant, contains an ~ 110-kb genomic deletion induced by T-DNA integration. FLC clade members were downregulated in the fly-1 mutants and FLY artificial microRNA knockdown line, whereas the level of the FLC antisense transcript COOLAIR was similar to that of the wild type. Our results identify FLY as a regulator that affects flowering time through upregulation of FLC clade members.

Knockdown of the DUF647 family member RUS4 impairs stamen development and pollen maturation in Arabidopsis

ROOT UV-B SENSITIVE4 (RUS4) encodes a Domain of Unknown Function647 (DUF647) protein, whose function is poorly understood. We have previously shown the artificial microRNA knockdown Arabidopsis RUS4 plants, referred to as amiR-RUS4, have severely reduced male fertility with a defect in anther dehiscence. Here, we show that amiR-RUS4 plants are also defective in pollen maturation and germination. Promoter-reporter analysis shows that RUS4 is highly expressed in tapetal layer, developing microspores, mature and germinating pollen, strongly suggesting its role in the process of pollen maturation. As the translational RUS4-GFP fusion protein has been localized to the chloroplasts where the first step of jasmonic acid (JA) biosynthesis takes place, leading to the hypothesis that RUS4 may be involved in JA-mediated stamen development. We show that expression of several JA metabolic genes increased markedly in flower buds of the amiR-RUS4 plants compared to that of the wild-type. We further show that transcript abundance of a clade of the JA-responsive MYB transcript factor genes, especially MYB108, reduced significantly in stamens of amiR-RUS4 plants relative to the wild-type; these MYB transcript factors have been shown to be required for JA-mediated stamen and pollen maturation. Our data suggest that RUS4 may play a role in coordinating anther dehiscence and pollen maturation by affecting the expression of JA-related genes.

Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato.

Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato. Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.